Cellobiohydrolase variants and polynucleotides encoding same

ABSTRACT

The present invention relates to variants of a parent cellobiohydrolase. The present invention also relates to polynucleotides encoding the cellobiohydrolase variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the cellobiohydrolase variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/US2011/030352, filed on Mar. 29, 2011, which claims priority fromU.S. provisional application Ser. No. 61/319,672, filed on Mar. 31,2010. The contents of these applications are fully incorporated hereinby reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under CooperativeAgreement DE-FC36-08GO18080 awarded by the Department of Energy. Thegovernment has certain rights in this invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to variants of a cellobiohydrolase,polynucleotides encoding the variants, methods of producing thevariants, and methods of using the variants.

2. Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently linked bybeta-1,4-bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the lignocellulose is converted tofermentable sugars, e.g., glucose, the fermentable sugars are easilyfermented by yeast into ethanol.

WO 2006/074005 discloses variants of a Hypocrea jecorinacellobiohydrolase II. Heinzelman et al., 2009, Proceedings of theNational Academy of Sciences USA 106: 5610-5615 discloses a family ofthermostable fungal cellulases created by structure-guidedrecombination. Heinzelman et al., 2009, Journal of Biological Chemistry284: 26229-26233 discloses a single mutation that contributes tostability of a fungal cellulase.

It would be advantageous in the art to improve the ability ofpolypeptides having cellobiohydrolase activity to enhance enzymaticdegradation of lignocellulosic feedstocks.

The present invention provides variants of a parent cellobiohydrolasewith improved properties compared to its parent.

SUMMARY OF THE INVENTION

The present invention relates to isolated variants of a parentcellobiohydrolase, comprising a substitution at one or more (several)positions corresponding to positions 254, 285, 286, 330, 342 and 360 ofSEQ ID NO: 2, wherein the variants have cellobiohydrolase activity. Inone aspect, the isolated variants further comprise a substitution at oneor more (several) positions corresponding to positions 245, 382, 420,437, and 440 of SEQ ID NO: 2.

The present invention also relates to isolated polynucleotides encodingthe variants; nucleic acid constructs, vectors, and host cellscomprising the polynucleotides; and methods of producing the variants.

The present invention also relates to plants comprising an isolatedpolynucleotide encoding such a variant having cellobiohydrolaseactivity.

The present invention also relates to methods of producing such avariant having cellobiohydrolase activity, comprising: (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe variant having cellobiohydrolase activity under conditions conducivefor production of the variant; and (b) recovering the variant.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of such a varianthaving cellobiohydrolase activity. In one aspect, the method furthercomprises recovering the degraded or converted cellulosic material.

The present invention also relates to methods of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of such a varianthaving cellobiohydrolase activity; (b) fermenting the saccharifiedcellulosic material of step (a) with one or more fermentingmicroorganisms to produce the fermentation product; and (c) recoveringthe fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore fermenting microorganisms, wherein the cellulosic material issaccharified with an enzyme composition in the presence of such avariant having cellobiohydrolase activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows a comparison of the residual activity for wild-typeAspergillus fumigatus family GH6A cellobiohydrolase and severalindividual variants of the Aspergillus fumigatus family GH6Acellobiohydrolase.

FIG. 1B shows a comparison of the residual activity for wild-typeAspergillus fumigatus family GH6A cellobiohydrolase and several combinedvariants of the Aspergillus fumigatus family GH6A cellobiohydrolase.

FIG. 2 shows a comparison of the hydrolytic activity for wild-typeAspergillus fumigatus family GH6A cellobiohydrolase and several combinedvariants of the Aspergillus fumigatus family GH6A cellobiohydrolase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated variants of a parentcellobiohydrolase, comprising a substitution at one or more (several)positions corresponding to positions 254, 285, 286, 330, 342, and 360 ofSEQ ID NO: 2, wherein the variants have cellobiohydrolase activity. Insome aspects, the variants have improved thermostability.

DEFINITIONS

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91), which catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain (Teeri, 1997, Crystalline cellulose degradation: New insightinto the function of cellobiohydrolases, Trends in Biotechnology 15:160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: whyso efficient on crystalline cellulose?, Biochem. Soc. Trans. 26:173-178). For purposes of the present invention, cellobiohydrolaseactivity is determined using a fluorescent disaccharide derivative4-methylumbelliferyl-β-D-lactoside according to the procedures describedby van Tilbeurgh et al., 1982, FEBS Letters 149: 152-156 and vanTilbeurgh and Claeyssens, 1985, FEBS Letters 187: 283-288.“Cellobiohydrolase activity” means hydrolytic activity of cellulosicmaterial catalyzed by a cellobiohydrolase.

Cellulolytic activity: The term “cellulolytic activity” means abiological activity that hydrolyzes a cellulosic material. The two basicapproaches for measuring cellulolytic activity include: (1) measuringthe total cellulolytic activity, and (2) measuring the individualcellulolytic activities (endoglucanases, cellobiohydrolases, andbeta-glucosidases) as reviewed in Zhang et al., Outlook for cellulaseimprovement: Screening and selection strategies, 2006, BiotechnologyAdvances 24: 452-481. Total cellulolytic activity is usually measuredusing insoluble substrates, including Whatman No. 1 filter paper,microcrystalline cellulose, bacterial cellulose, algal cellulose,cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No.1 filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-20mg of cellulolytic protein/g of cellulose in PCS for 3-7 days at 50-65°C. compared to a control hydrolysis without addition of cellulolyticprotein. Typical conditions are 1 ml reactions, washed or unwashed PCS,5% insoluble solids, 50 mM sodium acetate pH 5, 1 mM MnSO₄, 50-65° C.,72 hours, sugar analysis by AMINEX® HPX-87H column (Bio-RadLaboratories, Inc., Hercules, Calif., USA).

Endoglucanase: The term “endoglucanase” means anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4),which catalyses endohydrolysis of 1,4-beta-D-glycosidic linkages incellulose, cellulose derivatives (such as carboxymethyl cellulose andhydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3glucans such as cereal beta-D-glucans or xyloglucans, and other plantmaterial containing cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21), which catalyzes the hydrolysis ofterminal non-reducing beta-D-glucose residues with the release ofbeta-D-glucose. For purposes of the present invention, beta-glucosidaseactivity is determined according to the basic procedure described byVenturi et al., 2002, Extracellular beta-D-glucosidase from Chaetomiumthermophilum var. coprophilum: production, purification and somebiochemical properties, J. Basic Microbiol. 42: 55-66. One unit ofbeta-glucosidase is defined as 1.0 μmole of p-nitrophenol produced perminute at 40° C., pH 5 from 1 mM p-nitrophenyl-beta-D-glucopyranoside assubstrate in 100 mM sodium citrate containing 0.01% TWEEN® 20.

Cellulolytic enhancing activity: The term “cellulolytic enhancingactivity” means a biological activity catalyzed by GH61 polypeptide thatenhances the hydrolysis of a cellulosic material by enzyme havingcellulolytic activity. For purposes of the present invention,cellulolytic enhancing activity is determined by measuring the increasein reducing sugars or the increase of the total of cellobiose andglucose from the hydrolysis of a cellulosic material by cellulolyticenzyme under the following conditions: 1-50 mg of total protein/g ofcellulose in PCS, wherein total protein is comprised of 50-99.5% w/wcellulolytic protein and 0.5-50% w/w protein of a GH61 polypeptidehaving cellulolytic enhancing activity for 1-7 day at 50-65° C. comparedto a control hydrolysis with equal total protein loading withoutcellulolytic enhancing activity (1-50 mg of cellulolytic protein/g ofcellulose in PCS). In a preferred aspect, a mixture of CELLUCLAST® 1.5 L(Novozymes A/S, Bagsværd, Denmark) in the presence of 3% of totalprotein weight Aspergillus oryzae beta-glucosidase (recombinantlyproduced in Aspergillus oryzae according to WO 02/095014) or 3% of totalprotein weight Aspergillus fumigatus beta-glucosidase (recombinantlyproduced in Aspergillus oryzae as described in WO 2002/095014) ofcellulase protein loading is used as the source of the cellulolyticactivity.

The GH61 polypeptides having cellulolytic enhancing activity have atleast 20%, preferably at least 40%, more preferably at least 50%, morepreferably at least 60%, more preferably at least 70%, more preferablyat least 80%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 100% of the cellulolyticenhancing activity of the mature polypeptide of a GH61 polypeptide.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, more preferably at least 1.05-fold, more preferably at least1.10-fold, more preferably at least 1.25-fold, more preferably at least1.5-fold, more preferably at least 2-fold, more preferably at least3-fold, more preferably at least 4-fold, more preferably at least5-fold, even more preferably at least 10-fold, and most preferably atleast 20-fold.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696.

Family 6 glycoside hydrolase: The term “Family 6 glycoside hydrolase” or“Family GH6” or “GH6” means a polypeptide falling into the glycosidehydrolase Family 6 according to Henrissat B., 1991, supra, and HenrissatB., and Bairoch A., 1996, supra.

Xylan degrading activity: The terms “xylan degrading activity” or“xylanolytic activity” mean a biological activity that hydrolyzesxylan-containing material. The two basic approaches for measuringxylanolytic activity include: (1) measuring the total xylanolyticactivity, and (2) measuring the individual xylanolytic activities(endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, Recent progress in the assays of xylanolytic enzymes, 2006,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek,1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctionalbeta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. The most common total xylanolytic activity assay is basedon production of reducing sugars from polymeric 4-O-methylglucuronoxylan as described in Bailey, Biely, Poutanen, 1992,Interlaboratory testing of methods for assay of xylanase activity,Journal of Biotechnology 23(3): 257-270.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endo-hydrolysis of1,4-beta-D-xylosidic linkages in xylans. For purposes of the presentinvention, xylanase activity is determined using birchwood xylan assubstrate. One unit of xylanase is defined as 1.0 mmole of reducingsugar (measured in glucose equivalents as described by Lever, 1972, Anew reaction for colorimetric determination of carbohydrates, Anal.Biochem 47: 273-279) produced per minute during the initial period ofhydrolysis at 50° C., pH 5 from 2 g of birchwood xylan per liter assubstrate in 50 mM sodium acetate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides, to remove successive D-xylose residuesfrom the non-reducing termini. For purposes of the present invention,one unit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolproduced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyses the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20. One unit of acetylxylan esterase is definedas the amount of enzyme capable of releasing 1 μmole of p-nitrophenolateanion per minute at pH 5, 25° C.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl)group from an esterified sugar, which is usually arabinose in “natural”substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloylesterase is also known as ferulic acid esterase, hydroxycinnamoylesterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, orFAE-II. For purposes of the present invention, feruloyl esteraseactivity is determined using 0.5 mM p-nitrophenylferulate as substratein 50 mM sodium acetate pH 5.0. One unit of feruloyl esterase equals theamount of enzyme capable of releasing 1 μmole of p-nitrophenolate anionper minute at pH 5, 25° C.

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Cellulosic material: The cellulosic material can be any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, herbaceous material,agricultural residue, forestry residue, municipal solid waste, wastepaper, and pulp and paper mill residue (see, for example, Wiselogel etal., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.105-118, Taylor & Francis, Washington D.C.; Wyman, 1994, BioresourceTechnology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion ofLignocellulosics, in Advances in Biochemical Engineering/Biotechnology,T. Scheper, managing editor, Volume 65, pp. 23-40, Springer-Verlag, NewYork). It is understood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is lignocellulose.

In one aspect, the cellulosic material is herbaceous material. Inanother aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is forestry residue. In anotheraspect, the cellulosic material is municipal solid waste. In anotheraspect, the cellulosic material is waste paper. In another aspect, thecellulosic material is pulp and paper mill residue.

In another aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is corn fiber. In another aspect, thecellulosic material is corn cob. In another aspect, the cellulosicmaterial is orange peel. In another aspect, the cellulosic material isrice straw. In another aspect, the cellulosic material is wheat straw.In another aspect, the cellulosic material is switch grass. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is bagasse.

In another aspect, the cellulosic material is microcrystallinecellulose. In another aspect, the cellulosic material is bacterialcellulose. In another aspect, the cellulosic material is algalcellulose. In another aspect, the cellulosic material is cotton linter.In another aspect, the cellulosic material is amorphous phosphoric-acidtreated cellulose. In another aspect, the cellulosic material is filterpaper.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid.

Xylan-containing material: The term “xylan-containing material” meansany material comprising a plant cell wall polysaccharide containing abackbone of beta-(1-4)-linked xylose residues. Xylans of terrestrialplants are heteropolymers possessing a beta-(1-4)-D-xylopyranosebackbone, which is branched by short carbohydrate chains. They compriseD-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or variousoligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose,and D-glucose. Xylan-type polysaccharides can be divided into homoxylansand heteroxylans, which include glucuronoxylans,(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, andcomplex heteroxylans. See, for example, Ebringerova et al., 2005, Adv.Polym. Sci. 186: 1-67.

In the methods of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Variant: The term “variant” means a polypeptide having cellobiohydrolaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (several) positions. A substitutionmeans a replacement of an amino acid occupying a position with adifferent amino acid; a deletion means removal of an amino acidoccupying a position; and an insertion means adding 1-3 amino acidsadjacent to an amino acid occupying a position.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Wild-Type: The term “wild-type” (e.g., as used in wild-type protein,wild-type polypeptide, or wild-type cellobiohydrolase) describes theindicated polypeptide which as been expressed by a naturally occurringmicroorganism, such as a bacterium, yeast, or filamentous fungus foundin nature.

Parent or parent cellobiohydrolase: The term “parent” or “parentcellobiohydrolase” means a cellobiohydrolase to which an alteration ismade to produce a cellobiohydrolase variant of the present invention.The parent may be a naturally occurring (wild-type) polypeptide or avariant thereof. For instance, the parent polypeptide may be a variantof a wild-type polypeptide. A parent may also be an allelic variant,which is a polypeptide encoded by any of two or more alternative formsof a gene occupying the same chromosomal locus.

Isolated: The terms “isolated” and “purified” mean a polypeptide orpolynucleotide that is removed from at least one component with which itis naturally associated. For example, a variant or polypeptide may be atleast 1% pure, e.g., at least 5% pure, at least 10% pure, at least 20%pure, at least 40% pure, at least 60% pure, at least 80% pure, at least90% pure, and at least 95% pure, as determined by SDS-PAGE and apolynucleotide may be at least 1% pure, e.g., at least 5% pure, at least10% pure, at least 20% pure, at least 40% pure, at least 60% pure, atleast 80% pure, at least 90% pure, and at least 95% pure, as determinedby agarose electrophoresis.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 20 to 454 of SEQ ID NO: 2 based on theSigcleave program (von Heijne G., 1986, Nucleic Acids Res. 14:4683-4690) that predicts amino acids 1 to 19 of SEQ ID NO: 2 are asignal peptide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving cellobiohydrolase activity (e.g., cDNA or genomic DNA). In oneaspect, the mature polypeptide coding sequence is nucleotides 58 to 1710of SEQ ID NO: 1 based on the Sigcleave program (von Heijne G., 1986,supra) that predicts nucleotides 1 to 57 of SEQ ID NO: 1 encode a signalpeptide. In another aspect, the mature polypeptide coding sequence isthe cDNA sequence contained in nucleotides 58 to 1710 of SEQ ID NO: 1.

Sequence Identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the -nobrief option) is used as the percent identity andis calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the -nobriefoption) is used as the percent identity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Fragment: The term “fragment” means a polypeptide having one or more(several) amino acids deleted from the amino and/or carboxyl terminus ofa mature polypeptide; wherein the fragment has cellobiohydrolaseactivity. In one aspect, a fragment contains at least 370 amino acidresidues, at least 390 amino acid residues, and at least 410 amino acidresidues.

Subsequence: The term “subsequence” means a polynucleotide having one ormore (several) nucleotides deleted from the 5′- and/or 3′-end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having cellobiohydrolase activity. In one aspect, a subsequencecontains at least 1110 nucleotides, at least 1170 nucleotides, and atleast 1230 nucleotides.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of its polypeptideproduct. The boundaries of the coding sequence are generally determinedby an open reading frame, which usually begins with the ATG start codonor alternative start codons such as GTG and TTG and ends with a stopcodon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA,synthetic, or recombinant polynucleotide.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic cell. cDNA lacks intron sequences that may be presentin the corresponding genomic DNA. The initial, primary RNA transcript isa precursor to mRNA that is processed through a series of steps,including splicing, before appearing as mature spliced mRNA.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic. The term nucleic acid construct issynonymous with the term “expression cassette” when the nucleic acidconstruct contains the control sequences required for expression of acoding sequence of the present invention.

Control sequences: The term “control sequences” means all componentsnecessary for the expression of a polynucleotide encoding a variant ofthe present invention. Each control sequence may be native or foreign tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs the expression of the coding sequence.

Expression: The term “expression” includes any step involved in theproduction of the variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to additional nucleotides that provide for itsexpression.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, and the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved property: The term “improved property” means a characteristicassociated with a variant that is improved compared to the parent. Suchimproved properties include, but are not limited to, thermal activity,thermostability, pH activity, pH stability, substrate/cofactorspecificity, improved surface properties, product specificity, increasedstability or solubility in the presence of pretreated biomass, improvedstability under storage conditions, and chemical stability.

Improved thermostability: The term “improved thermostability” means avariant displaying retention of cellobiohydrolase activity after aperiod of incubation at elevated temperature relative to the parent,either in a buffer or under conditions such as those which exist duringproduct storage/transport or conditions similar to those that existduring industrial use of the variant. A variant may or may not displayan altered thermal activity profile relative to the parent. For example,a variant may have an improved ability to refold following incubation atan elevated temperature relative to the parent.

In one aspect, the thermostability of the variant havingcellobiohydrolase activity is at least 1.05-fold, e.g., at least1.1-fold, at least 1.5-fold, at least 1.8-fold, at least 2-fold, atleast 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, andat least 25-fold more thermostable than the parent when residualactivity following a 20 minute heat challenge at 67° C. in 100 mMNaCl-50 mM sodium acetate pH 5.0 buffer is compared using a biomasshydrolysis assay, in which an equivalent addition of either the varianthaving cellobiohydrolase activity or parent enzyme, expressed in mgprotein per gram cellulose, is added to PASC at a fixed temperature of50° C. at pH 5 for a total hydrolysis time of 30 minutes, at which pointbiomass hydrolysis is measured.

Conventions for Designation of Variants

For purposes of the present invention, the polypeptide disclosed in SEQID NO: 2 is used to determine the corresponding amino acid residue inanother cellobiohydrolase. The amino acid sequence of anothercellobiohydrolase is aligned with the polypeptide disclosed in SEQ IDNO: 2, and based on the alignment, the amino acid position numbercorresponding to any amino acid residue in the polypeptide disclosed inSEQ ID NO: 2 is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 3.0.0 or later.

Identification of the corresponding amino acid residue in anothercellobiohydrolase can be confirmed by an alignment of multiplepolypeptide sequences using “ClustalW” (Larkin et al., 2007,Bioinformatics 23: 2947-2948).

When the other enzyme has diverged from the polypeptide of SEQ ID NO: 2such that traditional sequence-based comparison fails to detect theirrelationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615),other pairwise sequence comparison algorithms can be used. Greatersensitivity in sequence-based searching can be attained using searchprograms that utilize probabilistic representations of polypeptidefamilies (profiles) to search databases. For example, the PSI-BLASTprogram generates profiles through an iterative database search processand is capable of detecting remote homologs (Atschul et al., 1997,Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can beachieved if the family or superfamily for the polypeptide has one ormore representatives in the protein structure databases. Programs suchas GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin andJones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementations of thesealgorithms can additionally be utilized to query structure databaseswith a structure of interest in order to discover possible structuralhomologs (e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the cellobiohydrolase variants of the present invention,the nomenclature described below is adapted for ease of reference. Theaccepted IUPAC single letter or three letter amino acid abbreviation isemployed.

Substitutions. For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of threonine with alanine atposition 226 is designated as “Thr226Ala” or “T226A”. Multiple mutationsare separated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or“G205R+S411F”, representing substitutions at positions 205 and 411 ofglycine (G) with arginine (R) and serine (S) with phenylalanine (F),respectively.

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position*. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*”or “G195*+S411*”.

Insertions. For an amino acid insertion, the following nomenclature isused: Original amino acid, position, original amino acid, inserted aminoacid. Accordingly the insertion of lysine after glycine at position 195is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple alterations. Variants comprising multiple alterations areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of tyrosine and glutamic acidfor arginine and glycine at positions 170 and 195, respectively.

Different alterations. Where different alterations can be introduced ata position, the different alterations are separated by a comma, e.g.,“Arg170Tyr,Glu” represents a substitution of arginine with tyrosine orglutamic acid at position 170. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala”designates the following variants:

“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and“Tyr167Ala+Arg170Ala”.

Parent Cellobiohydrolases

The parent cellobiohydrolase may be (a) a polypeptide having at least60% sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) apolypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, (ii) the cDNA sequence contained in the mature polypeptidecoding sequence of SEQ ID NO: 1, or (iii) the full-length complementarystrand of (i) or (ii); or (c) a polypeptide encoded by a polynucleotidehaving at least 60% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 1.

In a first aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 2 of at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which havecellobiohydrolase activity. In one aspect, the amino acid sequence ofthe parent differs by no more than ten amino acids, e.g., by no morethan five amino acids, four amino acids, three amino acids, two aminoacids, or one amino acid from the mature polypeptide of SEQ ID NO: 2.

In one aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 2. In another aspect, the parent comprises orconsists of the mature polypeptide of SEQ ID NO: 2. In another aspect,the parent comprises or consists of amino acids 20 to 454 of SEQ ID NO:2.

In an embodiment, the parent is a fragment of the mature polypeptide ofSEQ ID NO: 2 containing at least 370 amino acid residues, e.g., at least390 and at least 410 amino acid residues.

In another embodiment, the parent is an allelic variant of the maturepolypeptide of SEQ ID NO: 2.

In a second aspect, the parent is encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) the full-length complementary strandof (i) or (ii) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe amino acid sequence of SEQ ID NO: 2 or a fragment thereof, may beused to design nucleic acid probes to identify and clone DNA encoding aparent from strains of different genera or species according to methodswell known in the art. In particular, such probes can be used forhybridization with the genomic or cDNA of the genus or species ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least14, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other organisms may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1 or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleotide probe corresponding tothe polynucleotide shown in SEQ ID NO: 1, its full-length complementarystrand, or a subsequence thereof, under low to very high stringencyconditions. Molecules to which the probe hybridizes can be detectedusing, for example, X-ray film or any other detection means known in theart.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1. In another aspect, the nucleic acid probe isnucleotides 58 to 1710 of SEQ ID NO: 1. In another aspect, the nucleicacid probe is a polynucleotide that encodes the polypeptide of SEQ IDNO: 2 or a fragment thereof. In another aspect, the nucleic acid probeis SEQ ID NO: 1.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and either 25% formamide for very lowand low stringencies, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed three times each for15 minutes using 2×SSC, 0.2% SDS at 45° C. (very low stringency), 50° C.(low stringency), 55° C. (medium stringency), 60° C. (medium-highstringency), 65° C. (high stringency), or 70° C. (very high stringency).

For short probes that are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization andhybridization at about 5° C. to about 10° C. below the calculated T_(m)using the calculation according to Bolton and McCarthy (1962, Proc.Natl. Acad. Sci. USA 48: 1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA perml following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed once in 6×SCC plus0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5°C. to 10° C. below the calculated T_(m).

In a third aspect, the parent is encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, which encodes a polypeptide havingcellobiohydrolase activity. In one aspect, the mature polypeptide codingsequence is nucleotides 58 to 1710 of SEQ ID NO: 1. In an embodiment,the parent is encoded by a polynucleotide comprising or consisting ofSEQ ID NO: 1.

The parent may be obtained from microorganisms of any genus. Forpurposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a cell inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a bacterial cellobiohydrolase. For example, the parentmay be a gram-positive bacterial polypeptide such as a Bacillus,Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus,Oceanobacillus, Staphylococcus, Streptococcus, or Streptomycescellobiohydrolase, or a gram-negative bacterial polypeptide such as aCampylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasmacellobiohydrolase.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis cellobiohydrolase.

In another aspect, the parent is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus cellobiohydrolase.

In another aspect, the parent is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans cellobiohydrolase.

The parent may be a fungal cellobiohydrolase. For example, the parentmay be a yeast cellobiohydrolase such as a Candida, Kluyveromyces,Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiacellobiohydrolase. For example, the parent may be a filamentous fungalcellobiohydrolase such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria cellobiohydrolase.

In another aspect, the parent is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis cellobiohydrolase.

In another aspect, the parent is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus Fumigatus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa,Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viridecellobiohydrolase.

In another aspect, the parent is an Aspergillus cellobiohydrolase, suchas an Aspergillus fumigatus cellobiohydrolase, e.g., thecellobiohydrolase of SEQ ID NO: 2 or the mature polypeptide thereof.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc,) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. The polynucleotide encoding a parent may then bederived by similarly screening a genomic or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with a probe(s), the polynucleotide may beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

The parent may be a hybrid polypeptide in which a portion of onepolypeptide is fused at the N-terminus or the C-terminus of a portion ofanother polypeptide.

The parent also may be a fused polypeptide or cleavable fusionpolypeptide in which one polypeptide is fused at the N-terminus or theC-terminus of another polypeptide. A fused polypeptide is produced byfusing a polynucleotide encoding one polypeptide to a polynucleotideencoding another polypeptide. Techniques for producing fusionpolypeptides are known in the art, and include ligating the codingsequences encoding the polypeptides so that they are in frame and thatexpression of the fused polypeptide is under control of the samepromoter(s) and terminator. Fusion proteins may also be constructedusing intein technology in which fusions are createdpost-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawsonet al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Preparation of Variants

The present invention also relates to methods for obtaining a varianthaving cellobiohydrolase activity, comprising: (a) introducing into aparent cellobiohydrolase a substitution at one or more (several)positions corresponding to positions 254, 285, 286, 330, 342, and 360 ofthe mature polypeptide of SEQ ID NO: 2, wherein the variant hascellobiohydrolase activity; and (b) recovering the variant. In oneaspect, the method further comprises introducing into the parentcellobiohydrolase a substitution at one or more (several) positionscorresponding to positions 254, 285, 286, 330, 342, and 360 of SEQ IDNO: 2.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Site-directed mutagenesis is a technique in which one or more (several)mutations are created at one or more defined sites in a polynucleotideencoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests at theplasmid and the oligonucleotide is the same, permitting sticky ends ofthe plasmid and insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotideare synthesized and assembled upon photo-programmable microfluidicchips.

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCR implication.Polynucleotide subsequences may then be shuffled.

Variants

The present invention also provides variants of a parentcellobiohydrolase comprising a substitution at one or more (several)positions corresponding to positions 254, 285, 286, 330, 342, and 360 ofSEQ ID NO: 2, wherein the variant has cellobiohydrolase activity.

In an aspect, the variant has sequence identity of at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100%, to the amino acid sequence of the parentcellobiohydrolase.

In another aspect, the variant has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, suchas at least 96%, at least 97%, at least 98%, and at least 99%, but lessthan 100%, sequence identity to the mature polypeptide of SEQ ID NO: 2.

In one aspect, the variants of the present invention comprise 1-5substitutions, such as 1, 2, 3, 4, or 5 substitutions. In anotheraspect, the variants of the present invention comprise 1-3substitutions, such as 1, 2, or 3 substitutions.

In one aspect, a variant comprises a substitution at one or more(several) positions corresponding to positions 254, 285, 286, 330, 342,and 360 of SEQ ID NO: 2. In another aspect, the variant comprises orconsists of one or more (several) substitutions selected from C254L,L285I, G286Q, D330N, M342F, and S360G corresponding to SEQ ID NO: 2.

In one aspect, a variant comprises a substitution at any one positioncorresponding to positions 254, 285, 286, 330, 342, and 360 of SEQ IDNO: 2. In another aspect, the variant comprises or consists of onesubstitution selected from C254L, L285I, G286Q, D330N, M342F, and S360Gcorresponding to SEQ ID NO: 2.

In one aspect, the variant comprises or consists of a substitution at aposition corresponding to position 254 of SEQ ID NO: 2. In anotheraspect, the amino acid at a position corresponding to position 254 issubstituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Leu. Inanother aspect, the variant comprises or consists of the substitutionC254L corresponding to SEQ ID NO: 2.

In one aspect, the variant comprises or consists of a substitution at aposition corresponding to position 285 of SEQ ID NO: 2. In anotheraspect, the amino acid at a position corresponding to position 285 issubstituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ile. Inanother aspect, the variant comprises or consists of the substitutionL285I corresponding to SEQ ID NO: 2.

In one aspect, the variant comprises or consists of a substitution at aposition corresponding to position 286 of SEQ ID NO: 2. In anotheraspect, the amino acid at a position corresponding to position 286 issubstituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Gln. Inanother aspect, the variant comprises or consists of the substitutionG286Q corresponding to SEQ ID NO: 2.

In one aspect, the variant comprises or consists of a substitution at aposition corresponding to position 286 of SEQ ID NO: 2 together with asubstitution at a position corresponding to position 286 of SEQ ID NO:2. In another aspect, the amino acids at the positions corresponding topositions 285 and 286 are substituted with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,preferably with Ile and Gln, for positions 285 and 286, respectively. Inanother aspect, the variant comprises or consists of the substitutionL285I together with the substitution G286Q corresponding to SEQ ID NO:2.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 330 of SEQ ID NO: 2. In anotheraspect, the amino acid at a position corresponding to position 330 issubstituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Asn. Inanother aspect, the variant comprises or consists of the substitutionD330N corresponding to SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 342 of SEQ ID NO: 2. In anotheraspect, the amino acid at a position corresponding to position 342 issubstituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Phe. Inanother aspect, the variant comprises or consists of the substitutionM342F corresponding to SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 360 of SEQ ID NO: 2. In anotheraspect, the amino acid at a position corresponding to position 360 issubstituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Gly. Inanother aspect, the variant comprises or consists of the substitutionS360G corresponding to SEQ ID NO: 2.

In one aspect, the variant comprises or consists of substitutions at anytwo positions corresponding to positions 254, 285, 286, 330, 342, and360 of SEQ ID NO: 2 (e.g., 285 and 286), such as those described above.In one aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 254 and 285 of SEQ ID NO: 2, suchas those described above. In another aspect, the variant comprises orconsists of substitutions at positions corresponding to positions 254and 286, such as those described above. In another aspect, the variantcomprises or consists of substitutions at positions corresponding topositions 254 and 330, such as those described above. In another aspect,the variant comprises or consists of substitutions at positionscorresponding to positions 254 and 342, such as those described above.In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 254 and 360, such as thosedescribed above. In another aspect, the variant comprises or consists ofsubstitutions at positions corresponding to positions 285 and 286, suchas those described above. In another aspect, the variant comprises orconsists of substitutions at positions corresponding to positions 285and 330, such as those described above. In another aspect, the variantcomprises or consists of substitutions at positions corresponding topositions 285 and 342, such as those described above. In another aspect,the variant comprises or consists of substitutions at positionscorresponding to positions 285 and 360, such as those described above.In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 286 and 330, such as thosedescribed above. In another aspect, the variant comprises or consists ofsubstitutions at positions corresponding to positions 286 and 342, suchas those described above. In another aspect, the variant comprises orconsists of substitutions at positions corresponding to positions 286and 360, such as those described above.

In one aspect, the variant comprises or consists of two substitutionsselected from C254L, L285I, G286Q, D330N, M342F, and S360G correspondingto SEQ ID NO: 2. In one aspect, the variant comprises or consists ofsubstitutions C254L+L285I. In another aspect, the variant comprises orconsists of substitutions C254L+G286Q. In another aspect, the variantcomprises or consists of substitutions C254L+D330N. In another aspect,the variant comprises or consists of substitutions C254L+M342F. Inanother aspect, the variant comprises or consists of substitutionsC254L+S360G. In another aspect, the variant comprises or consists ofsubstitutions L285I+G286Q. In another aspect, the variant comprises orconsists of substitutions L285I+D330N. In another aspect, the variantcomprises or consists of substitutions L285I+M342F. In another aspect,the variant comprises or consists of substitutions L285I+S360G. Inanother aspect, the variant comprises or consists of substitutionsG286Q+D330N. In another aspect, the variant comprises or consists ofsubstitutions G286Q+M342F. In another aspect, the variant comprises orconsists of substitutions G286Q+S360G.

In one aspect, the variant comprises or consists of substitutions at anythree positions corresponding to positions 254, 285, 286, 330, 342, and360 of SEQ ID NO: 2, such as those described above. In one aspect, thevariant comprises or consists of substitutions at positionscorresponding to positions 254, 285, and 286 of SEQ ID NO: 2, such asthose described above. In another aspect, the variant comprises orconsists of substitutions at positions corresponding to positions 254,285, and 330, such as those described above. In another aspect, thevariant comprises or consists of substitutions at positionscorresponding to positions 254, 285, and 342, such as those describedabove. In another aspect, the variant comprises or consists ofsubstitutions at positions corresponding to positions 254, 285, and 360,such as those described above. In another aspect, the variant comprisesor consists of substitutions at positions corresponding to positions254, 286, and 330, such as those described above. In another aspect, thevariant comprises or consists of substitutions at positionscorresponding to positions 254, 286, and 342, such as those describedabove. In another aspect, the variant comprises or consists ofsubstitutions at positions corresponding to positions 254, 286, and 360,such as those described above. In another aspect, the variant comprisesor consists of substitutions at positions corresponding to positions254, 330, and 342, such as those described above. In another aspect, thevariant comprises or consists of substitutions at positionscorresponding to positions 254, 330, and 360, such as those describedabove. In another aspect, the variant comprises or consists ofsubstitutions at positions corresponding to positions 254, 342, and 360,such as those described above. In another aspect, the variant comprisesor consists of substitutions at positions corresponding to positions285, 286, and 330, such as those described above. In another aspect, thevariant comprises or consists of substitutions at positionscorresponding to positions 285, 286, and 342, such as those describedabove. In another aspect, the variant comprises or consists ofsubstitutions at positions corresponding to positions 285, 286, and 360,such as those described above. In another aspect, the variant comprisesor consists of substitutions at positions corresponding to positions285, 330, and 342, such as those described above. In another aspect, thevariant comprises or consists of substitutions at positionscorresponding to positions 285, 330, and 360, such as those describedabove. In another aspect, the variant comprises or consists ofsubstitutions at positions corresponding to positions 285, 342, and 360,such as those described above. In another aspect, the variant comprisesor consists of substitutions at positions corresponding to positions286, 330, and 342, such as those described above. In another aspect, thevariant comprises or consists of substitutions at positionscorresponding to positions 286, 330, and 360, such as those describedabove. In another aspect, the variant comprises or consists ofsubstitutions at positions corresponding to positions 286, 342, and 360,such as those described above. In another aspect, the variant comprisesor consists of substitutions at positions corresponding to positions330, 342, and 360, such as those described above.

In another aspect, the variant comprises or consists of threesubstitutions selected from C254L, L285I, G286Q, D330N, M342F, and S360Gcorresponding to SEQ ID NO: 2. In one aspect, the variant comprises orconsists of the substitutions C254L+L285I+G286Q. In another aspect, thevariant comprises or consists of the substitutions C254L+L285I+D330N. Inanother aspect, the variant comprises or consists of the substitutionsC254L+L285I+M342F. In another aspect, the variant comprises or consistsof the substitutions C254L+L285I+S360G. In another aspect, the variantcomprises or consists of the substitutions C254L+G286Q+D330N. In anotheraspect, the variant comprises or consists of the substitutionsC254L+G286Q+M342F. In another aspect, the variant comprises or consistsof the substitutions C254L+G286Q+S360G. In another aspect, the variantcomprises or consists of the substitutions C254L+D330N+M342F. In anotheraspect, the variant comprises or consists of the substitutionsC254L+D330N+S360G. In another aspect, the variant comprises or consistsof the substitutions C254L+M342F+S360G. In another aspect, the variantcomprises or consists of the substitutions L285I+G286Q+D330N. In anotheraspect, the variant comprises or consists of the substitutionsL285I+G286Q+M342F. In another aspect, the variant comprises or consistsof the substitutions L285I+G286Q+S360G. In another aspect, the variantcomprises or consists of the substitutions L285I+D330N+M342F. In anotheraspect, the variant comprises or consists of the substitutionsL285I+D330N+S360G. In another aspect, the variant comprises or consistsof the substitutions L285I+M342F+S360G. In another aspect, the variantcomprises or consists of the substitutions G286Q+D330N+M342F. In anotheraspect, the variant comprises or consists of the substitutionsG286Q+D330N+S360G. In another aspect, the variant comprises or consistsof the substitutions G286Q+M342F+S360G. In another aspect, the variantcomprises or consists of the substitutions D330N+M342F+S360G.

In one aspect, the variant comprises or consists of substitutions at anyfour positions corresponding to positions 254, 285, 286, 330, 342, and360 of SEQ ID NO: 2, such as those described above. In one aspect, thevariant comprises or consists of substitutions at positionscorresponding to positions 254, 285, 286, and 330 of SEQ ID NO: 2, suchas those described above. In another aspect, the variant comprises orconsists of substitutions at positions corresponding to positions 254,285, 286, and 342, such as those described above. In another aspect, thevariant comprises or consists of substitutions at positionscorresponding to positions 254, 285, 330, and 342, such as thosedescribed above. In another aspect, the variant comprises or consists ofsubstitutions at positions corresponding to positions 254, 286, 330, and342, such as those described above. In another aspect, the variantcomprises or consists of substitutions at positions corresponding topositions 285, 286, 330, and 342, such as those described above. Inanother aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 254, 285, 286, and 360, such asthose described above. In another aspect, the variant comprises orconsists of substitutions at positions corresponding to positions 254,285, 330, and 360, such as those described above. In another aspect, thevariant comprises or consists of substitutions at positionscorresponding to positions 254, 286, 330, and 360, such as thosedescribed above. In another aspect, the variant comprises or consists ofsubstitutions at positions corresponding to positions 285, 286, 330, and360, such as those described above. In another aspect, the variantcomprises or consists of substitutions at positions corresponding topositions 254, 285, 342, and 360, such as those described above. Inanother aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 254, 286, 342, and 360, such asthose described above. In another aspect, the variant comprises orconsists of substitutions at positions corresponding to positions 285,286, 342, and 360, such as those described above. In another aspect, thevariant comprises or consists of substitutions at positionscorresponding to positions 254, 330, 342, and 360, such as thosedescribed above. In another aspect, the variant comprises or consists ofsubstitutions at positions corresponding to positions 285, 330, 342, and360, such as those described above. In another aspect, the variantcomprises or consists of substitutions at positions corresponding topositions 286, 330, 342, and 360, such as those described above.

In another aspect, the variant comprises or consists of foursubstitutions selected from C254L, L285I, G286Q, D330N, M342F, and S360Gcorresponding to SEQ ID NO: 2. In one aspect, the variant comprises orconsists of the substitutions C254L+L285I+G286Q+D330N. In anotheraspect, the variant comprises or consists of the substitutionsC254L+L285I+G286Q+M342F. In another aspect, the variant comprises orconsists of the substitutions C254L+L285I+D330N+M342F. In anotheraspect, the variant comprises or consists of the substitutionsC254L+G286Q+D330N+M342F. In another aspect, the variant comprises orconsists of the substitutions L285I+G286Q+D330N+M342F. In anotheraspect, the variant comprises or consists of the substitutionsC254L+L285I+G286Q+S360G. In another aspect, the variant comprises orconsists of the substitutions C254L+L285I+D330N+S360G. In anotheraspect, the variant comprises or consists of the substitutionsC254L+G286Q+D330N+S360G. In another aspect, the variant comprises orconsists of the substitutions L285I+G286Q+D330N+S360G. In anotheraspect, the variant comprises or consists of the substitutionsC254L+L285I+M342F+S360G. In another aspect, the variant comprises orconsists of the substitutions C254L+G286Q+M342F+S360G. In anotheraspect, the variant comprises or consists of the substitutionsL285I+G286Q+M342F+S360G. In another aspect, the variant comprises orconsists of the substitutions C254L+D330N+M342F+S360G. In anotheraspect, the variant comprises or consists of the substitutionsL285I+D330N+M342F+S360G. In another aspect, the variant comprises orconsists of the substitutions G286Q+D330N+M342F+S360G.

In one aspect, the variant comprises or consists of substitutions at anyfive positions corresponding to positions 254, 285, 286, 330, 342, and360 of SEQ ID NO: 2, such as those described above. In one aspect, thevariant comprises or consists of substitutions at positionscorresponding to positions 285, 286, 330, 342, and 360 of SEQ ID NO: 2,such as those described above. In another aspect, the variant comprisesor consists of substitutions at positions corresponding to positions254, 286, 330, 342, and 360, such as those described above. In anotheraspect, the variant comprises or consists of substitutions at positionscorresponding to positions 254, 285, 330, 342, and 360, such as thosedescribed above. In another aspect, the variant comprises or consists ofsubstitutions at positions corresponding to positions 254, 285, 286,342, and 360, such as those described above. In another aspect, thevariant comprises or consists of substitutions at positionscorresponding to positions 254, 285, 286, 330, and 360, such as thosedescribed above. In another aspect, the variant comprises or consists ofsubstitutions at positions corresponding to positions 254, 285, 286,330, and 342, such as those described above.

In another aspect, the variant comprises or consists of fivesubstitutions selected from C254L, L285I, G286Q, D330N, M342F, and S360Gcorresponding to SEQ ID NO: 2. In one aspect, the variant comprises orconsists of the substitutions L285I+G286Q+D330N+M342F+S360G. In anotheraspect, the variant comprises or consists of the substitutionsC254L+G286Q+D330N+M342F+S360G. In another aspect, the variant comprisesor consists of the substitutions C254L+L285I+D330N+M342F+S360G. Inanother aspect, the variant comprises or consists of the substitutionsC254L+L285I+G286Q+M342F+S360G. In another aspect, the variant comprisesor consists of the substitutions C254L+L285I+G286Q+D330N+S360G. Inanother aspect, the variant comprises or consists of the substitutionsC254L+L285I+G286Q+D330N+M342F.

In another aspect, the variant comprises or consists of substitutionscorresponding to positions 254, 285, 286, 330, 342, and 360 of SEQ IDNO: 2, such as those described above. In another aspect, the variantcomprises or consists of the substitutionsC254L+L285I+G286Q+D330N+M342F+S360G corresponding to SEQ ID NO: 2.

The variants may further comprise an alteration at one or more (several)other positions. In one aspect, the variants of the present inventioncomprise 1-5 further alterations, such as 1, 2, 3, 4, or 5 alterations.In another aspect, the variants of the present invention furthercomprise 1-5 substitutions, such as 1, 2, 3, 4, or 5 substitutions.

In one aspect, a variant comprises or consists of a substitution at oneor more (several) positions corresponding to positions 245, 382, 420,437, and 440 of SEQ ID NO: 2. In another aspect, the variant comprisesor consists of one or more (several) substitutions selected from A245S,G382C, L420I, T437Q, and Q440C corresponding to SEQ ID NO: 2.

In one aspect, a variant comprises or consists of a substitution at anyone position corresponding to positions 245, 382, 420, 437, and 440 ofSEQ ID NO: 2. In another aspect, the variant comprises or consists ofone substitution selected from A245S, G382C, L420I, T437Q, and Q440Ccorresponding to SEQ ID NO: 2.

In one aspect, the variant further comprises a substitution at aposition corresponding to position 245 of SEQ ID NO: 2. In anotheraspect, the amino acid at a position corresponding to position 245 issubstituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ser. Inanother aspect, the variant further comprises or consists of thesubstitution A245S corresponding to SEQ ID NO: 2.

In one aspect, the variant further comprises a substitution at aposition corresponding to position 382 of SEQ ID NO: 2. In anotheraspect, the amino acid at a position corresponding to position 382 issubstituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Cys. Inanother aspect, the variant further comprises or consists of thesubstitution G382C corresponding to SEQ ID NO: 2.

In one aspect, the variant further comprises a substitution at aposition corresponding to position 420 of SEQ ID NO: 2. In anotheraspect, the amino acid at a position corresponding to position 420 issubstituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ile. Inanother aspect, the variant further comprises or consists of thesubstitution L420I corresponding to SEQ ID NO: 2.

In one aspect, the variant further comprises a substitution at aposition corresponding to position 437 of SEQ ID NO: 2. In anotheraspect, the amino acid at a position corresponding to position 437 issubstituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Gln. Inanother aspect, the variant further comprises or consists of thesubstitution T437Q corresponding to SEQ ID NO: 2.

In one aspect, the variant further comprises a substitution at aposition corresponding to position 440 of SEQ ID NO: 2. In anotheraspect, the amino acid at a position corresponding to position 440 issubstituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Cys. Inanother aspect, the variant further comprises or consists of thesubstitution Q440C corresponding to SEQ ID NO: 2. In one aspect, thevariant further comprises or consists of the substitutions G382C andQ440C corresponding to SEQ ID NO: 2.

In one aspect, the variant further comprises or consists ofsubstitutions at any two positions corresponding to positions 245, 382,420, 437, and 440 of SEQ ID NO: 2, such as those described above. In oneaspect, the variant further comprises or consists of a substitution atpositions corresponding to positions 420 and 437 of SEQ ID NO: 2, suchas those described above. In another aspect, the variant furthercomprises or consists of substitutions at positions corresponding topositions 245 and 420, such as those described above. In another aspect,the variant further comprises or consists of substitutions at positionscorresponding to positions 382 and 420, such as those described above.In another aspect, the variant further comprises or consists ofsubstitutions at positions corresponding to positions 420 and 440, suchas those described above. In another aspect, the variant furthercomprises or consists of a substitution at positions corresponding topositions 245 and 437, such as those described above. In another aspect,the variant further comprises or consists of substitutions at positionscorresponding to positions 382 and 437, such as those described above.In another aspect, the variant further comprises or consists ofsubstitutions at positions corresponding to positions 437 and 440, suchas those described above. In another aspect, the variant furthercomprises or consists of a substitution at positions corresponding topositions 245 and 382, such as those described above. In another aspect,the variant further comprises or consists of a substitution at positionscorresponding to positions 245 and 440, such as those described above.In another aspect, the variant further comprises or consists of asubstitution at positions corresponding to positions 382 and 440, suchas those described above.

In another aspect, the variant further comprises or consists of any twosubstitutions selected from A245S, G382C, L420I, T437Q, and Q440Ccorresponding to SEQ ID NO: 2. In one aspect, the variant furthercomprises or consists of the substitutions L420I+T437Q corresponding toSEQ ID NO: 2. In another aspect, the variant further comprises orconsists of the substitutions A245S+L420I. In another aspect, thevariant further comprises or consists of the substitutions G382C+L420I.In another aspect, the variant further comprises or consists of thesubstitutions L420I+Q440C. In another aspect, the variant furthercomprises or consists of the substitutions A245S+T437Q. In anotheraspect, the variant further comprises or consists of the substitutionsG382C+T437Q. In another aspect, the variant further comprises orconsists of the substitutions T437Q+Q440C. In another aspect, thevariant further comprises or consists of the substitutions A245S+G382C.In another aspect, the variant further comprises or consists of thesubstitutions A245S+Q440C. In another aspect, the variant furthercomprises or consists of the substitutions G382C+Q440C.

In one aspect, the variant further comprises or consists ofsubstitutions at any three positions corresponding to positions 245,382, 420, 437, and 440 of SEQ ID NO: 2, such as those described above.In one aspect, the variant further comprises or consists of asubstitution at positions corresponding to positions 245, 420, and 437of SEQ ID NO: 2, such as those described above. In another aspect, thevariant further comprises or consists of a substitution at positionscorresponding to positions 382, 420, and 437, such as those describedabove. In another aspect, the variant further comprises or consists of asubstitution at positions corresponding to positions 420, 437, and 440,such as those described above. In another aspect, the variant furthercomprises or consists of a substitution at positions corresponding topositions 245, 382, and 420 such as those described above. In anotheraspect, the variant further comprises or consists of a substitution atpositions corresponding to positions 245, 420, and 440, such as thosedescribed above. In another aspect, the variant further comprises orconsists of a substitution at positions corresponding to positions 382,420, and 440, such as those described above. In another aspect, thevariant further comprises or consists of a substitution at positionscorresponding to positions 245, 382, and 437, such as those describedabove. In another aspect, the variant further comprises or consists of asubstitution at positions corresponding to positions 245, 437, and 440,such as those described above. In another aspect, the variant furthercomprises or consists of a substitution at positions corresponding topositions 382, 437, and 440, such as those described above. In anotheraspect, the variant further comprises or consists of a substitution atpositions corresponding to positions 245, 382, and 440, such as thosedescribed above.

In another aspect, the variant further comprises or consists of anythree substitutions selected from A245S, G382C, L420I, T437Q, and Q440Ccorresponding to SEQ ID NO: 2. In one aspect, the variant furthercomprises or consists of the substitutions A245S+L420I+T437Qcorresponding to SEQ ID NO: 2. In another aspect, the variant furthercomprises or consists of the substitutions G382C+L420I+T437Q. In anotheraspect, the variant further comprises or consists of the substitutionsL420I+T437Q+Q440C. In another aspect, the variant further comprises orconsists of the substitutions A245S+G382C+L420I. In another aspect, thevariant further comprises or consists of the substitutionsA245S+L420I+Q440C. In another aspect, the variant further comprises orconsists of the substitutions G382C+L420I+Q440C. In another aspect, thevariant further comprises or consists of the substitutionsA245S+G382C+T437Q. In another aspect, the variant further comprises orconsists of the substitutions A245S+T437Q+Q440C. In another aspect, thevariant further comprises or consists of the substitutionsG382C+T437Q+Q440C. In another aspect, the variant further comprises orconsists of the substitutions A245S+G382C+Q440C.

In one aspect, the variant further comprises or consists ofsubstitutions at any four positions corresponding to positions 245, 382,420, 437, and 440 of SEQ ID NO: 2, such as those described above. In oneaspect, the variant further comprises or consists of a substitution atpositions corresponding to positions 245, 382, 437, and 440 of themature polypeptide of SEQ ID NO: 2, such as those described above. Inanother aspect, the variant further comprises or consists of asubstitution at positions corresponding to positions 245, 382, 420, and440, such as those described above. In another aspect, the variantfurther comprises or consists of a substitution at positionscorresponding to positions 382, 420, 437, and 440, such as thosedescribed above. In another aspect, the variant further comprises orconsists of a substitution at positions corresponding to positions 245,420, 437, and 440, such as those described above. In another aspect, thevariant further comprises or consists of a substitution at positionscorresponding to positions 245, 382, 420, and 437, such as thosedescribed above.

In another aspect, the variant further comprises or consists of any foursubstitutions selected from A245S, G382C, L420I, T437Q, and Q440Ccorresponding to SEQ ID NO: 2. In one aspect, the variant furthercomprises or consists of the substitutions A245S+G382C+T437Q+Q440C ofthe corresponding to SEQ ID NO: 2. In another aspect, the variantfurther comprises or consists of the substitutionsA245S+G382C+L420I+Q440C. In another aspect, the variant furthercomprises or consists of the substitutions G382C+L420I+T437Q+Q440C. Inanother aspect, the variant further comprises or consists of thesubstitutions A245S+L420I+T437Q+Q440C. In another aspect, the variantfurther comprises or consists of the substitutionsA245S+G382C+L420I+T437Q.

In one aspect, the variant further comprises or consists of asubstitution at positions corresponding to positions 245, 382, 420, 437,and 440 of SEQ ID NO: 2, such as those described above. In one aspect,the variant further comprises or consists of the substitutionsA245S+G382C+L420I+T437Q+Q440C corresponding to SEQ ID NO: 2.

Essential amino acids in a parent can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for cellobiohydrolase activity to identify aminoacid residues that are critical to the activity of the molecule. Seealso, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The activesite of the cellobiohydrolase or other biological interaction can alsobe determined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities ofessential amino acids can also be inferred from analysis of identitieswith polypeptides that are related to the parent.

The variants may consist of 370 to 450 amino acids, e.g., 370 to 379,380 to 389, 390 to 399, 400 to 409, 410 to 419, and 420 to 440 aminoacids.

The invention also encompasses polypeptides having cellobiohydrolaseactivity, wherein one or more (several) positions of the amino acidsequence differ from corresponding positions 254, 285, 286, 330, 342,and 360 of SEQ ID NO: 2. The variant aspects described herein alsoembrace similar aspects related to polypeptides having cellobiohydrolaseactivity.

Polynucleotides

The present invention also relates to isolated polynucleotides thatencode any of the variants of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more (several) control sequences that direct theexpression of the coding sequence in a suitable host cell underconditions compatible with the control sequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of a variant. Manipulation of the polynucleotide prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter sequence, which is recognized bya host cell for expression of the polynucleotide. The promoter sequencecontains transcriptional control sequences that mediate the expressionof the variant. The promoter may be any nucleic acid sequence that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, E. colilac operon, Streptomyces coelicolor agarase gene (dagA), and prokaryoticbeta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci.USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983,Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are describedin “Useful proteins from recombinant bacteria” in Gilbert et al., 1980,Scientific American 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are the promoters obtained from the genes for Aspergillusnidulans acetamidase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger orAspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKAamylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Fusarium oxysporum trypsin-like protease (WO96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusariumvenenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900),Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase,Trichoderma reesei beta-glucosidase, Trichoderma reeseicellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II,Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanaseIV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, aswell as the NA2-tpi promoter (a modified promoter including a geneencoding a neutral alpha-amylase in Aspergilli in which the untranslatedleader has been replaced by an untranslated leader from a gene encodingtriose phosphate isomerase in Aspergilli; non-limiting examples includemodified promoters including the gene encoding neutral alpha-amylase inAspergillus niger in which the untranslated leader has been replaced byan untranslated leader from the gene encoding triose phosphate isomerasein Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated,and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, which is recognized by a host cell to terminate transcription.The terminator sequence is operably linked to the 3′-terminus of thepolynucleotide encoding the variant. Any terminator that is functionalin the host cell may be used.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger alpha-glucosidase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′-terminus ofthe polynucleotide encoding the variant. Any leader sequence that isfunctional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding region naturally linked in translation reading frame withthe segment of the coding region that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding region that is foreign to the coding sequence. Theforeign signal peptide coding region may be required where the codingsequence does not naturally contain a signal peptide coding region.Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to enhancesecretion of the variant. However, any signal peptide coding region thatdirects the expressed variant into the secretory pathway of a host cellmay be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that encodesa propeptide positioned at the N-terminus of a variant. The resultantpolypeptide is known as a proenzyme or propolypeptide (or a zymogen insome cases). A propolypeptide is generally inactive and can be convertedto an active polypeptide by catalytic or autocatalytic cleavage of thepropeptide from the propolypeptide. The propeptide coding region may beobtained from the genes for Bacillus subtilis alkaline protease (aprE),Bacillus subtilis neutral protease (nprT), Myceliophthora thermophilalaccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, andSaccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide regions are present at theN-terminus of a variant, the propeptide region is positioned next to theN-terminus of the variant and the signal peptide region is positionednext to the N-terminus of the propeptide region.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the variant relative to the growth ofthe host cell. Examples of regulatory systems are those that cause theexpression of the gene to be turned on or off in response to a chemicalor physical stimulus, including the presence of a regulatory compound.Regulatory systems in prokaryotic systems include the lac, tac, and trpoperator systems. In yeast, the ADH2 system or GAL1 system may be used.In filamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the variant would be operably linkedwith the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more (several) convenientrestriction sites to allow for insertion or substitution of thepolynucleotide encoding the variant at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the polynucleotide. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vector maybe a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more (several) selectable markersthat permit easy selection of transformed, transfected, transduced, orthe like cells. A selectable marker is a gene the product of whichprovides for biocide or viral resistance, resistance to heavy metals,prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacilluslicheniformis or Bacillus subtilis, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofidentity to the corresponding target sequence to enhance the probabilityof homologous recombination. The integrational elements may be anysequence that is homologous with the target sequence in the genome ofthe host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleotide sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means anucleotide sequence that enables a plasmid or vector to replicate invivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into the host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra) to obtain isolated variants.

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or more(several) control sequences that direct the production of a variant ofthe present invention. A construct or vector comprising a polynucleotideis introduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thevariant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any gram-positive or gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell, including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell, including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Mol. Gen. Genet. 168: 111-115), by using competent cells (see, e.g.,Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), by electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or byconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may, forinstance, be effected by protoplast transformation (see, e.g., Hanahan,1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Doweret al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNAinto a Streptomyces cell may, for instance, be effected by protoplasttransformation and electroporation (see, e.g., Gong et al., 2004, FoliaMicrobiol. (Praha) 49: 399-405), by conjugation (see, e.g., Mazodier etal., 1989, J. Bacteriol. 171: 3583-3585), or by transduction (see, e.g.,Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). Theintroduction of DNA into a Pseudomonas cell may, for instance, beeffected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol.Methods 64: 391-397) or by conjugation (see, e.g., Pinedo and Smets,2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA intoa Streptococcus cell may, for instance, be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), by protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-2070, by electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, F. A., Passmore, S. M., andDavenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9,1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocaflimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus Fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell. In one aspect, the hostcell is an Aspergillus, e.g., Aspergillus oryzae.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023 and Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol.153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a host cell of the present invention underconditions suitable for the expression of the variant; and (b)recovering the variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants. These detection methods may include use ofspecific antibodies, formation of an enzyme product, or disappearance ofan enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the variant.

The variant may be recovered by methods known in the art. For example,the variant may be recovered from the nutrient medium by conventionalprocedures including, but not limited to, collection, centrifugation,filtration, extraction, spray-drying, evaporation, or precipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989) to obtainisolated variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing a variant is used as asource of the variant.

Compositions

The present invention also relates to compositions comprising a variantof the present invention. Preferably, the compositions are enriched insuch a variant. The term “enriched” means that the cellobiohydrolaseactivity of the composition has been increased, e.g., with an enrichmentfactor of 1.1.

The composition may comprise a variant as the major enzymatic component,e.g., a mono-component composition. Alternatively, the composition maycomprise multiple enzymatic activities, such as an aminopeptidase,amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase,lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase,peroxidase, phytase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transglutaminase, or xylanase. The additional enzyme(s)may be produced, for example, by a microorganism belonging to the genusAspergillus, e.g., Aspergillus aculeatus, Aspergillus awamori,Aspergillus foetidus, Aspergillus Fumigatus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae;Fusarium, e.g., Fusarium bactridioides, Fusarium cerealis, Fusariumcrookwellense, Fusarium culmorum, Fusarium graminearum, Fusariumgraminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusariumsarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusariumtrichothecioides, or Fusarium venenatum; Humicola, e.g., Humicolainsolens or Humicola lanuginosa; or Trichoderma, e.g., Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Forinstance, the composition may be in the form of a granulate or amicrogranulate. The variant may be stabilized in accordance with methodsknown in the art.

Processing of Cellulosic Material

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a variant havingcellobiohydrolase activity of the present invention. In a preferredaspect, the method further comprises recovering the degraded orconverted cellulosic material.

The present invention also relates to methods of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a variant havingcellobiohydrolase activity of the present invention; (b) fermenting thesaccharified cellulosic material with one or more (several) fermentingmicroorganisms to produce the fermentation product; and (c) recoveringthe fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of avariant having cellobiohydrolase activity of the present invention. In apreferred aspect, the fermenting of the cellulosic material produces afermentation product. In another preferred aspect, the method furthercomprises recovering the fermentation product from the fermentation.

The methods of the present invention can be used to saccharify acellulosic material to fermentable sugars and convert the fermentablesugars to many useful substances, e.g., fuel, potable ethanol, and/orfermentation products (e.g., acids, alcohols, ketones, gases, and thelike). The production of a desired fermentation product from cellulosicmaterial typically involves pretreatment, enzymatic hydrolysis(saccharification), and fermentation.

The processing of cellulosic material according to the present inventioncan be accomplished using processes conventional in the art. Moreover,the methods of the present invention can be implemented using anyconventional biomass processing apparatus configured to operate inaccordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and cofermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and fermentation (HHCF); anddirect microbial conversion (DMC). SHF uses separate process steps tofirst enzymatically hydrolyze cellulosic material to fermentable sugars,e.g., glucose, cellobiose, cellotriose, and pentose sugars, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the cofermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF involves a separate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more(several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd, L. R.,Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbialcellulose utilization: Fundamentals and biotechnology, Microbiol. Mol.Biol. Reviews 66: 506-577). It is understood herein that any methodknown in the art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof, can be usedin the practicing the methods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996,Enhancement of enzymatic cellulose hydrolysis using a novel type ofbioreactor with intensive stirring induced by electromagnetic field,Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor typesinclude: fluidized bed, upflow blanket, immobilized, and extruder typereactors for hydrolysis and/or fermentation.

Pretreatment. In practicing the methods of the present invention, anypretreatment process known in the art can be used to disrupt plant cellwall components of cellulosic material (Chandra et al., 2007, Substratepretreatment: The key to effective enzymatic hydrolysis oflignocellulosics? Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe andZacchi, 2007, Pretreatment of lignocellulosic materials for efficientbioethanol production, Adv. Biochem. Engin./Biotechnol. 108: 41-65;Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility oflignocellulosic biomass, Bioresource Technol. 100: 10-18; Mosier et al.,2005, Features of promising technologies for pretreatment oflignocellulosic biomass, Bioresource Technol. 96: 673-686; Taherzadehand Karimi, 2008, Pretreatment of lignocellulosic wastes to improveethanol and biogas production: A review, Int. J. of Mol. Sci. 9:1621-1651; Yang and Wyman, 2008, Pretreatment: the key to unlockinglow-cost cellulosic ethanol, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, pre-soaking, wetting, washing, or conditioning prior topretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, and gamma irradiationpretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, cellulosic material is heatedto disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. Cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably done at 140-230° C., more preferably 160-200° C., and mostpreferably 170-190° C., where the optimal temperature range depends onany addition of a chemical catalyst. Residence time for the steampretreatment is preferably 1-15 minutes, more preferably 3-12 minutes,and most preferably 4-10 minutes, where the optimal residence timedepends on temperature range and any addition of a chemical catalyst.Steam pretreatment allows for relatively high solids loadings, so thatcellulosic material is generally only moist during the pretreatment. Thesteam pretreatment is often combined with an explosive discharge of thematerial after the pretreatment, which is known as steam explosion, thatis, rapid flashing to atmospheric pressure and turbulent flow of thematerial to increase the accessible surface area by fragmentation (Duffand Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi,2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent ApplicationNo. 20020164730). During steam pretreatment, hemicellulose acetyl groupsare cleaved and the resulting acid autocatalyzes partial hydrolysis ofthe hemicellulose to monosaccharides and oligosaccharides. Lignin isremoved to only a limited extent.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 3% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762).

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Examples of suitable chemicalpretreatment processes include, for example, dilute acid pretreatment,lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX),ammonia percolation (APR), and organosolv pretreatments.

In dilute acid pretreatment, cellulosic material is mixed with diluteacid, typically H₂SO₄, and water to form a slurry, heated by steam tothe desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91:179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to, limepretreatment, wet oxidation, ammonia percolation (APR), and ammoniafiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium carbonate, sodium hydroxide,or ammonia at low temperatures of 85-150° C. and residence times from 1hour to several days (Wyman et al., 2005, Bioresource Technol. 96:1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO2006/110891, WO 2006/11899, WO 2006/11900, and WO 2006/110901 disclosepretreatment methods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed at preferably 1-40% drymatter, more preferably 2-30% dry matter, and most preferably 5-20% drymatter, and often the initial pH is increased by the addition of alkalisuch as sodium carbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion), can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-100°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). AFEX pretreatment results in the depolymerization ofcellulose and partial hydrolysis of hemicellulose. Lignin-carbohydratecomplexes are cleaved.

Organosolv pretreatment delignifies cellulosic material by extractionusing aqueous ethanol (40-60% ethanol) at 160-200° C. for 30-60 minutes(Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan et al., 2006,Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl. Biochem.Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose isremoved.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as anacid treatment, and more preferably as a continuous dilute and/or mildacid treatment. The acid is typically sulfuric acid, but other acids canalso be used, such as acetic acid, citric acid, nitric acid, phosphoricacid, tartaric acid, succinic acid, hydrogen chloride, or mixturesthereof. Mild acid treatment is conducted in the pH range of preferably1-5, more preferably 1-4, and most preferably 1-3. In one aspect, theacid concentration is in the range from preferably 0.01 to 20 wt % acid,more preferably 0.05 to 10 wt % acid, even more preferably 0.1 to 5 wt %acid, and most preferably 0.2 to 2.0 wt % acid. The acid is contactedwith cellulosic material and held at a temperature in the range ofpreferably 160-220° C., and more preferably 165-195° C., for periodsranging from seconds to minutes to, e.g., 1 second to 60 minutes.

In another aspect, pretreatment is carried out as an ammonia fiberexplosion step (AFEX pretreatment step).

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, cellulosic material is present during pretreatment inamounts preferably between 10-80 wt %, more preferably between 20-70 wt%, and most preferably between 30-60 wt %, such as around 50 wt %. Thepretreated cellulosic material can be unwashed or washed using anymethod known in the art, e.g., washed with water.

Mechanical Pretreatment: The term “mechanical pretreatment” refers tovarious types of grinding or milling (e.g., dry milling, wet milling, orvibratory ball milling).

Physical Pretreatment: The term “physical pretreatment” refers to anypretreatment that promotes the separation and/or release of cellulose,hemicellulose, and/or lignin from cellulosic material. For example,physical pretreatment can involve irradiation (e.g., microwaveirradiation), steaming/steam explosion, hydrothermolysis, andcombinations thereof.

Physical pretreatment can involve high pressure and/or high temperature(steam explosion). In one aspect, high pressure means pressure in therange of preferably about 300 to about 600 psi, more preferably about350 to about 550 psi, and most preferably about 400 to about 500 psi,such as around 450 psi. In another aspect, high temperature meanstemperatures in the range of about 100 to about 300° C., preferablyabout 140 to about 235° C. In a preferred aspect, mechanicalpretreatment is performed in a batch-process, steam gun hydrolyzersystem that uses high pressure and high temperature as defined above,e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.

Combined Physical and Chemical Pretreatment: Cellulosic material can bepretreated both physically and chemically. For instance, thepretreatment step can involve dilute or mild acid treatment and hightemperature and/or pressure treatment. The physical and chemicalpretreatments can be carried out sequentially or simultaneously, asdesired. A mechanical pretreatment can also be included.

Accordingly, in a preferred aspect, cellulosic material is subjected tomechanical, chemical, or physical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996,Pretreatment of biomass, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212; Ghosh and Singh, 1993, Physicochemical and biologicaltreatments for enzymatic/microbial conversion of cellulosic biomass,Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreatinglignocellulosic biomass: a review, in Enzymatic Conversion of Biomassfor Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P.,eds., ACS Symposium Series 566, American Chemical Society, Washington,D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,1999, Ethanol production from renewable resources, in Advances inBiochemical Engineering/Biotechnology, Scheper, T., ed., Springer-VerlagBerlin Heidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996,Fermentation of lignocellulosic hydrolysates for ethanol production,Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990,Production of ethanol from lignocellulosic materials: State of the art,Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification.

In the hydrolysis step, also known as saccharification, the cellulosicmaterial, e.g., pretreated, is hydrolyzed to break down cellulose andalternatively also hemicellulose to fermentable sugars, such as glucose,cellobiose, xylose, xylulose, arabinose, mannose, galactose, and/orsoluble oligosaccharides. The hydrolysis is performed enzymatically byan enzyme composition in the presence of a variant havingcellobiohydrolase activity of the present invention. The composition canfurther comprise one or more (several) hemicellulolytic or xylandegrading enzymes. The enzymes of the compositions can also be addedsequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In a preferred aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the cellulosic material (substrate), e.g.,pretreated, is fed gradually to, for example, an enzyme containinghydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 96 hours, more preferably about 16 to about 72 hours,and most preferably about 24 to about 48 hours. The temperature is inthe range of preferably about 25° C. to about 70° C., more preferablyabout 30° C. to about 65° C., and more preferably about 40° C. to 60°C., in particular about 50° C. The pH is in the range of preferablyabout 3 to about 8, more preferably about 3.5 to about 7, and mostpreferably about 4 to about 6, in particular about pH 5. The dry solidscontent is in the range of preferably about 5 to about 50 wt %, morepreferably about 10 to about 40 wt %, and most preferably about 20 toabout 30 wt %.

The enzyme composition preferably comprises enzymes having cellulolyticactivity and/or xylan degrading activity. In one aspect, the enzymecomposition comprises one or more (several) cellulolytic enzymes. Inanother aspect, the enzyme composition comprises one or more (several)xylan degrading enzymes. In another aspect, the enzyme compositioncomprises one or more (several) cellulolytic enzymes and one or more(several) xylan degrading enzymes.

The one or more (several) cellulolytic enzymes are preferably selectedfrom the group consisting of an endoglucanase, a cellobiohydrolase, anda beta-glucosidase. The one or more (several) xylan degrading enzymesare preferably selected from the group consisting of a xylanase, anacetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

In another aspect, the enzyme composition further or even furthercomprises a polypeptide having cellulolytic enhancing activity (see, forexample, WO 2005/074647, WO 2005/074656, and WO 2007/089290). In anotheraspect, the enzyme composition may further or even further comprise oneor more (several) additional enzyme activities to improve thedegradation of the cellulose-containing material. Preferred additionalenzymes are hemicellulases (e.g., alpha-D-glucuronidases,alpha-L-arabinofuranosidases, endo-mannanases, beta-mannosidases,alpha-galactosidases, endo-alpha-L-arabinanases, beta-galactosidases),carbohydrate-esterases (e.g., acetyl-xylan esterases, acetyl-mannanesterases, ferulic acid esterases, coumaric acid esterases, glucuronoylesterases), pectinases, proteases, ligninolytic enzymes (e.g., laccases,manganese peroxidases, lignin peroxidases, H₂O₂-producing enzymes,oxidoreductases), expansins, swollenins, or mixtures thereof. In themethods of the present invention, the additional enzyme(s) can be addedprior to or during fermentation, e.g., during saccharification or duringor after propagation of the fermenting microorganism(s).

One or more (several) components of the enzyme composition may bewild-type proteins, recombinant proteins, or a combination of wild-typeproteins and recombinant proteins. For example, one or more (several)components may be native proteins of a cell, which is used as a hostcell to express recombinantly one or more (several) other components ofthe enzyme composition. One or more (several) components of the enzymecomposition may be produced as monocomponents, which are then combinedto form the enzyme composition. The enzyme composition may be acombination of multicomponent and monocomponent protein preparations.

The enzymes used in the methods of the present invention may be in anyform suitable for use in the processes described herein, such as, forexample, a crude fermentation broth with or without cells removed, acell lysate with or without cellular debris, a semi-purified or purifiedenzyme preparation, or a host cell as a source of the enzymes. Theenzyme composition may be a dry powder or granulate, a non-dustinggranulate, a liquid, a stabilized liquid, or a stabilized protectedenzyme. Liquid enzyme preparations may, for instance, be stabilized byadding stabilizers such as a sugar, a sugar alcohol or another polyol,and/or lactic acid or another organic acid according to establishedprocesses.

The optimum amounts of the enzymes and variants having cellobiohydrolaseactivity depend on several factors including, but not limited to, themixture of component cellulolytic enzymes, the cellulosic substrate, theconcentration of cellulosic substrate, the pretreatment(s) of thecellulosic substrate, temperature, time, pH, and inclusion of fermentingorganism (e.g., yeast for Simultaneous Saccharification andFermentation).

In a preferred aspect, an effective amount of cellulolytic enzyme(s) tocellulosic material is about 0.5 to about 50 mg, preferably at about 0.5to about 40 mg, more preferably at about 0.5 to about 25 mg, morepreferably at about 0.75 to about 20 mg, more preferably at about 0.75to about 15 mg, even more preferably at about 0.5 to about 10 mg, andmost preferably at about 2.5 to about 10 mg per g of cellulosicmaterial.

In another preferred aspect, an effective amount of a variant havingcellobiohydrolase activity to cellulosic material is about 0.01 to about50.0 mg, preferably about 0.01 to about 40 mg, more preferably about0.01 to about 30 mg, more preferably about 0.01 to about 20 mg, morepreferably about 0.01 to about 10 mg, more preferably about 0.01 toabout 5 mg, more preferably at about 0.025 to about 1.5 mg, morepreferably at about 0.05 to about 1.25 mg, more preferably at about0.075 to about 1.25 mg, more preferably at about 0.1 to about 1.25 mg,even more preferably at about 0.15 to about 1.25 mg, and most preferablyat about 0.25 to about 1.0 mg per g of cellulosic material.

In another preferred aspect, an effective amount of a variant havingcellobiohydrolase activity to cellulolytic enzyme(s) is about 0.005 toabout 1.0 g, preferably at about 0.01 to about 1.0 g, more preferably atabout 0.15 to about 0.75 g, more preferably at about 0.15 to about 0.5g, more preferably at about 0.1 to about 0.5 g, even more preferably atabout 0.1 to about 0.5 g, and most preferably at about 0.05 to about 0.2g per g of cellulolytic enzyme(s).

The enzymes can be derived or obtained from any suitable origin,including, bacterial, fungal, yeast, plant, or mammalian origin. Theterm “obtained” means herein that the enzyme may have been isolated froman organism that naturally produces the enzyme as a native enzyme. Theterm “obtained” also means herein that the enzyme may have been producedrecombinantly in a host organism employing methods described herein,wherein the recombinantly produced enzyme is either native or foreign tothe host organism or has a modified amino acid sequence, e.g., havingone or more (several) amino acids that are deleted, inserted and/orsubstituted, i.e., a recombinantly produced enzyme that is a mutantand/or a fragment of a native amino acid sequence or an enzyme producedby nucleic acid shuffling processes known in the art. Encompassed withinthe meaning of a native enzyme are natural variants and within themeaning of a foreign enzyme are variants obtained recombinantly, such asby site-directed mutagenesis or shuffling.

A polypeptide having cellulolytic enzyme activity or xylan degradingactivity may be a bacterial polypeptide. For example, the polypeptidemay be a gram positive bacterial polypeptide such as a Bacillus,Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, or Oceanobacilluspolypeptide having cellulolytic enzyme activity or xylan degradingactivity, or a Gram negative bacterial polypeptide such as an E. coli,Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium,Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide havingcellulolytic enzyme activity or xylan degrading activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having cellulolytic enzyme activity or xylandegrading activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having cellulolyticenzyme activity or xylan degrading activity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingcellulolytic enzyme activity or xylan degrading activity.

The polypeptide having cellulolytic enzyme activity or xylan degradingactivity may also be a fungal polypeptide, and more preferably a yeastpolypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia polypeptide having cellulolytic enzymeactivity or xylan degrading activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having cellulolyticenzyme activity or xylan degrading activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having cellulolyticenzyme activity or xylan degrading activity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum,Penicillium purpurogenum, Phanerochaete chrysosporium, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspora, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,Trichoderma viride, or Trichophaea saccata polypeptide havingcellulolytic enzyme activity or xylan degrading activity.

Chemically modified or protein engineered mutants of polypeptides havingcellulolytic enzyme activity or xylan degrading activity may also beused.

One or more (several) components of the enzyme composition may be arecombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticproteins may also be prepared by purifying such a protein from afermentation broth.

Examples of commercial cellulolytic protein preparations suitable foruse in the present invention include, for example, CELLIC™ Ctec(Novozymes A/S), CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188 (NovozymesA/S), CELLUZYME™ (Novozymes A/S), CEREFLO™ (Novozymes A/S), andULTRAFLO™ (Novozymes A/S), ACCELERASE™ (Genencor Int.), LAMINEX™(Genencor Int.), SPEZYME™ CP (Genencor Int.), ROHAMENT™ 7069 W (RöhmGmbH), FIBREZYME® LDI (Dyadic International, Inc.), FIBREZYME® LBR(Dyadic International, Inc.), or VISCOSTAR® 150L (Dyadic International,Inc.). The cellulase enzymes are added in amounts effective from about0.001 to about 5.0 wt % of solids, more preferably from about 0.025 toabout 4.0 wt % of solids, and most preferably from about 0.005 to about2.0 wt % of solids. The cellulase enzymes are added in amounts effectivefrom about 0.001 to about 5.0 wt % of solids, more preferably from about0.025 to about 4.0 wt % of solids, and most preferably from about 0.005to about 2.0 wt % of solids.

Examples of bacterial endoglucanases that can be used in the methods ofthe present invention, include, but are not limited to, an Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the methods of thepresent invention, include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263; GENBANK™accession no. M15665); Trichoderma reesei endoglucanase II (Saloheimo,et al., 1988, Gene 63:11-22; GENBANK™ accession no. M19373); Trichodermareesei endoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol.64: 555-563; GENBANK™ accession no. AB003694); and Trichoderma reeseiendoglucanase V (Saloheimo et al., 1994, Molecular Microbiology 13:219-228; GENBANK™ accession no. Z33381); Aspergillus aculeatusendoglucanase (Ooi et al., 1990, Nucleic Acids Research 18: 5884);Aspergillus kawachii endoglucanase (Sakamoto et al., 1995, CurrentGenetics 27: 435-439); Erwinia carotovara endoglucanase (Saarilahti etal., 1990, Gene 90: 9-14); Fusarium oxysporum endoglucanase (GENBANK™accession no. L29381); Humicola grisea var. thermoidea endoglucanase(GENBANK™ accession no. AB003107); Melanocarpus albomyces endoglucanase(GENBANK™ accession no. MAL515703); Neurospora crassa endoglucanase(GENBANK™ accession no. XM_(—)324477); Humicola insolens endoglucanaseV; Myceliophthora thermophila CBS 117.65 endoglucanase; basidiomyceteCBS 495.95 endoglucanase; basidiomycete CBS 494.95 endoglucanase;Thielavia terrestris NRRL 8126 CEL6B endoglucanase; Thielavia terrestrisNRRL 8126 CEL6C endoglucanase); Thielavia terrestris NRRL 8126 CEL7Cendoglucanase; Thielavia terrestris NRRL 8126 CEL7E endoglucanase;Thielavia terrestris NRRL 8126 CEL7F endoglucanase; Cladorrhinumfoecundissimum ATCC 62373 CEL7A endoglucanase; and Trichoderma reeseistrain No. VTT-D-80133 endoglucanase (GENBANK™ accession no. M15665).

Examples of cellobiohydrolases useful in the methods of the presentinvention include, but are not limited to, Trichoderma reeseicellobiohydrolase I; Trichoderma reesei cellobiohydrolase II; Humicolainsolens cellobiohydrolase I, Myceliophthora thermophilacellobiohydrolase II, Thielavia terrestris cellobiohydrolase II (CEL6A),Chaetomium thermophilum cellobiohydrolase I, and Chaetomium thermophilumcellobiohydrolase II.

Examples of beta-glucosidases useful in the methods of the presentinvention include, but are not limited to, Aspergillus oryzaebeta-glucosidase; Aspergillus fumigatus beta-glucosidase; Penicilliumbrasilianum IBT 20888 beta-glucosidase; Aspergillus nigerbeta-glucosidase; and Aspergillus aculeatus beta-glucosidase.

The Aspergillus oryzae polypeptide having beta-glucosidase activity canbe obtained according to WO 2002/095014. The Aspergillus fumigatuspolypeptide having beta-glucosidase activity can be obtained accordingto WO 2005/047499. The Penicillium brasilianum polypeptide havingbeta-glucosidase activity can be obtained according to WO 2007/019442.The Aspergillus niger polypeptide having beta-glucosidase activity canbe obtained according to Dan et al., 2000, J. Biol. Chem. 275:4973-4980. The Aspergillus aculeatus polypeptide having beta-glucosidaseactivity can be obtained according to Kawaguchi et al., 1996, Gene 173:287-288.

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is the Aspergillus oryzae beta-glucosidase variant BGfusion protein or the Aspergillus oryzae beta-glucosidase fusion proteinobtained according to WO 2008/057637.

Other endoglucanases, cellobiohydrolases, and beta-glucosidases aredisclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in EP 495,257, EP 531,315, EP 531,372, WO 89/09259, WO94/07998, WO 95/24471, WO 96/11262, WO 96/29397, WO 96/034108, WO97/14804, WO 98/08940, WO 98/012307, WO 98/13465, WO 98/015619, WO98/015633, WO 98/028411, WO 99/06574, WO 99/10481, WO 99/025846, WO99/025847, WO 99/031255, WO 2000/009707, WO 2002/050245, WO2002/0076792, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO2008/008793, U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,457,046, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,691,178, U.S.Pat. No. 5,763,254, and U.S. Pat. No. 5,776,757.

In the methods of the present invention, any polypeptide havingcellulolytic enhancing activity can be used.

In a first aspect, the polypeptide having cellulolytic enhancingactivity comprises the following motifs:

[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] and[FW]-[TF]-K-[AIV],

wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5contiguous positions, and X(4) is any amino acid at 4 contiguouspositions.

The polypeptide comprising the above-noted motifs may further comprise:

H-X(1,2)-G-P-X(3)-[YW]-[AILMV],

[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV], or

H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV],

wherein X is any amino acid, X(1,2) is any amino acid at 1 position or 2contiguous positions, X(3) is any amino acid at 3 contiguous positions,and X(2) is any amino acid at 2 contiguous positions. In the abovemotifs, the accepted IUPAC single letter amino acid abbreviation isemployed.

In a preferred aspect, the polypeptide having cellulolytic enhancingactivity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV]. In anotherpreferred aspect, the isolated polypeptide having cellulolytic enhancingactivity further comprises [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV]. Inanother preferred aspect, the polypeptide having cellulolytic enhancingactivity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV].

In a second aspect, the polypeptide having cellulolytic enhancingactivity comprises the following motif:

[ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A-[HNQ],

wherein x is any amino acid, x(4,5) is any amino acid at 4 or 5contiguous positions, and x(3) is any amino acid at 3 contiguouspositions. In the above motif, the accepted IUPAC single letter aminoacid abbreviation is employed.

Examples of polypeptides having cellulolytic enhancing activity usefulin the methods of the present invention include, but are not limited to,polypeptides having cellulolytic enhancing activity from Thielaviaterrestris (WO 2005/074647); polypeptides having cellulolytic enhancingactivity from Thermoascus aurantiacus (WO 2005/074656); and polypeptideshaving cellulolytic enhancing activity from Trichoderma reesei (WO2007/089290).

Examples of commercial xylan degrading enzyme preparations suitable foruse in the present invention include, for example, SHEARZYME™ (NovozymesA/S), CELLIC™ Htec (Novozymes A/S), VISCOZYME® (Novozymes A/S),ULTRAFLO® (Novozymes A/S), PULPZYME® HC (Novozymes A/S), MULTIFECT®Xylanase (Genencor), ECOPULP® TX-200A (AB Enzymes), HSP 6000 Xylanase(DSM), DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™ 740L.(Biocatalysts Limit, Wales, UK), and DEPOL™ 762P (Biocatalysts Limit,Wales, UK).

Examples of xylanases useful in the methods of the present inventioninclude, but are not limited to, Aspergillus aculeatus xylanase(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus xylanases (WO2006/078256), and Thielavia terrestris NRRL 8126 xylanases (WO2009/079210).

Examples of beta-xylosidases useful in the methods of the presentinvention include, but are not limited to, Trichoderma reeseibeta-xylosidase (UniProtKB/TrEMBL accession number Q92458), Talaromycesemersonii (SwissProt accession number Q8×212), and Neurospora crassa(SwissProt accession number Q7SOW4).

Examples of acetylxylan esterases useful in the methods of the presentinvention include, but are not limited to, Hypocrea jecorina acetylxylanesterase (WO 2005/001036), Neurospora crassa acetylxylan esterase(UniProt accession number q7s259), Thielavia terrestris NRRL 8126acetylxylan esterase (WO 2009/042846), Chaetomium globosum acetylxylanesterase (Uniprot accession number Q2GWX4), Chaetomium gracileacetylxylan esterase (GeneSeqP accession number AAB82124), Phaeosphaerianodorum acetylxylan esterase (Uniprot accession number Q0UHJ1), andHumicola insolens DSM 1800 acetylxylan esterase (WO 2009/073709).

Examples of ferulic acid esterases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800feruloyl esterase (WO 2009/076122), Neurospora crassa feruloyl esterase(UniProt accession number Q9HGR3), and Neosartorya fischeri feruloylesterase (UniProt Accession number A1D9T4).

Examples of arabinofuranosidases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800arabinofuranosidase (WO 2009/073383) and Aspergillus nigerarabinofuranosidase (GeneSeqP accession number AAR94170).

Examples of alpha-glucuronidases useful in the methods of the presentinvention include, but are not limited to, Aspergillus clavatusalpha-glucuronidase (UniProt accession number alcc12), Trichodermareesei alpha-glucuronidase (Uniprot accession number Q99024),Talaromyces emersonii alpha-glucuronidase (UniProt accession numberQ8×211), Aspergillus niger alpha-glucuronidase (Uniprot accession numberQ96WX9), Aspergillus terreus alpha-glucuronidase (SwissProt accessionnumber Q0CJP9), and Aspergillus fumigatus alpha-glucuronidase (SwissProtaccession number Q4WW45).

The enzymes and proteins used in the methods of the present inventionmay be produced by fermentation of the above-noted microbial strains ona nutrient medium containing suitable carbon and nitrogen sources andinorganic salts, using procedures known in the art (see, e.g., Bennett,J. W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, AcademicPress, CA, 1991). Suitable media are available from commercial suppliersor may be prepared according to published compositions (e.g., incatalogues of the American Type Culture Collection). Temperature rangesand other conditions suitable for growth and enzyme production are knownin the art (see, e.g., Bailey, J. E., and Ollis, D. F., BiochemicalEngineering Fundamentals, McGraw-Hill Book Company, NY, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme. Fermentation may, therefore,be understood as comprising shake flask cultivation, or small- orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the enzymeto be expressed or isolated. The resulting enzymes produced by themethods described above may be recovered from the fermentation mediumand purified by conventional procedures.

Fermentation.

The fermentable sugars obtained from the hydrolyzed cellulosic materialcan be fermented by one or more (several) fermenting microorganismscapable of fermenting the sugars directly or indirectly into a desiredfermentation product. “Fermentation” or “fermentation process” refers toany fermentation process or any process comprising a fermentation step.Fermentation processes also include fermentation processes used in theconsumable alcohol industry (e.g., beer and wine), dairy industry (e.g.,fermented dairy products), leather industry, and tobacco industry. Thefermentation conditions depend on the desired fermentation product andfermenting organism and can easily be determined by one skilled in theart.

In the fermentation step, sugars, released from cellulosic material as aresult of the pretreatment and enzymatic hydrolysis steps, are fermentedto a product, e.g., ethanol, by a fermenting organism, such as yeast.Hydrolysis (saccharification) and fermentation can be separate orsimultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be C₆ and/or C₅ fermenting organisms, or a combinationthereof. Both C₆ and C₅ fermenting organisms are well known in the art.Suitable fermenting microorganisms are able to ferment, i.e., convert,sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose,galactose, or oligosaccharides, directly or indirectly into the desiredfermentation product.

Examples of bacterial and fungal fermenting organisms producing ethanolare described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69:627-642.

Examples of fermenting microorganisms that can ferment C₆ sugars includebacterial and fungal organisms, such as yeast. Preferred yeast includesstrains of the Saccharomyces spp., preferably Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment C₅ sugars includebacterial and fungal organisms, such as yeast. Preferred C₅ fermentingyeast include strains of Pichia, preferably Pichia stipitis, such asPichia stipitis CBS 5773; strains of Candida, preferably Candidaboidinii, Candida brassicae, Candida sheatae, Candida diddensii, Candidapseudotropicalis, or Candida utilis.

Other fermenting organisms include strains of Zymomonas, such asZymomonas mobilis; Hansenula, such as Hansenula anomala; Kluyveromyces,such as K. fragilis; Schizosaccharomyces, such as S. pombe; and E. coli,especially E. coli strains that have been genetically modified toimprove the yield of ethanol.

In a preferred aspect, the yeast is a Saccharomyces spp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae. In another morepreferred aspect, the yeast is Saccharomyces distaticus. In another morepreferred aspect, the yeast is Saccharomyces uvarum. In anotherpreferred aspect, the yeast is a Kluyveromyces. In another morepreferred aspect, the yeast is Kluyveromyces marxianus. In another morepreferred aspect, the yeast is Kluyveromyces fragilis. In anotherpreferred aspect, the yeast is a Candida. In another more preferredaspect, the yeast is Candida boidinii. In another more preferred aspect,the yeast is Candida brassicae. In another more preferred aspect, theyeast is Candida diddensii. In another more preferred aspect, the yeastis Candida pseudotropicalis. In another more preferred aspect, the yeastis Candida utilis. In another preferred aspect, the yeast is aClavispora. In another more preferred aspect, the yeast is Clavisporalusitaniae. In another more preferred aspect, the yeast is Clavisporaopuntiae. In another preferred aspect, the yeast is a Pachysolen. Inanother more preferred aspect, the yeast is Pachysolen tannophilus. Inanother preferred aspect, the yeast is a Pichia. In another morepreferred aspect, the yeast is a Pichia stipitis. In another preferredaspect, the yeast is a Bretannomyces. In another more preferred aspect,the yeast is Bretannomyces clausenii (Philippidis, G. P., 1996,Cellulose bioconversion technology, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212).

Bacteria that can efficiently ferment hexose and pentose to ethanolinclude, for example, Zymomonas mobilis and Clostridium thermocellum(Philippidis, 1996, supra).

In a preferred aspect, the bacterium is a Zymomonas. In a more preferredaspect, the bacterium is Zymomonas mobilis. In another preferred aspect,the bacterium is a Clostridium. In another more preferred aspect, thebacterium is Clostridium thermocellum.

Commercially available yeast suitable for ethanol production includes,e.g., ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI™(available from Fleischmann's Yeast, USA), SUPERSTART™ and THERMOSACC™fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM™ AFTand XR (available from NABC—North American Bioproducts Corporation, GA,USA), GERT STRAND™ (available from Gert Strand AB, Sweden), and FERMIOL™(available from DSM Specialties).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (cofermentation) (Chen and Ho, 1993, Cloning andimproving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TAL1 genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Saccharomyces cerevisiae. In another preferred aspect, thegenetically modified fermenting microorganism is Zymomonas mobilis. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Escherichia coli. In another preferred aspect, thegenetically modified fermenting microorganism is Klebsiella oxytoca. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Kluyveromyces sp.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedlignocellulose or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, such as about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., inparticular about 32° C. or 50° C., and at about pH 3 to about pH 8, suchas around pH 4-5, 6, or 7.

In a preferred aspect, the yeast and/or another microorganism is appliedto the degraded cellulosic material and the fermentation is performedfor about 12 to about 96 hours, such as typically 24-60 hours. In apreferred aspect, the temperature is preferably between about 20° C. toabout 60° C., more preferably about 25° C. to about 50° C., and mostpreferably about 32° C. to about 50° C., in particular about 32° C. or50° C., and the pH is generally from about pH 3 to about pH 7,preferably around pH 4-7. However, some fermenting organisms, e.g.,bacteria, have higher fermentation temperature optima. Yeast or anothermicroorganism is preferably applied in amounts of approximately 10⁵ to10¹², preferably from approximately 10⁷ to 10¹⁰, especiallyapproximately 2×10⁸ viable cell count per ml of fermentation broth.Further guidance in respect of using yeast for fermentation can be foundin, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which ishereby incorporated by reference.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe methods of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation products: A fermentation product can be any substancederived from the fermentation. The fermentation product can be, withoutlimitation, an alcohol (e.g., arabinitol, butanol, ethanol, glycerol,methanol, 1,3-propanediol, sorbitol, and xylitol); an organic acid(e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citricacid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaricacid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionicacid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid,oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); aketone (e.g., acetone); an amino acid (e.g., aspartic acid, glutamicacid, glycine, lysine, serine, and threonine); and a gas (e.g., methane,hydrogen (H₂), carbon dioxide (CO₂), and carbon monoxide (CO)). Thefermentation product can also be protein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is arabinitol. In another more preferred aspect, the alcohol isbutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is glycerol. In another morepreferred aspect, the alcohol is methanol. In another more preferredaspect, the alcohol is 1,3-propanediol. In another more preferredaspect, the alcohol is sorbitol. In another more preferred aspect, thealcohol is xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira, M.M., and Jonas, R., 2002, The biotechnological production of sorbitol,Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and Singh, D.,1995, Processes for fermentative production of xylitol—a sugarsubstitute, Process Biochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi,N. and Blaschek, H. P., 2003, Production of acetone, butanol and ethanolby Clostridium beijerinckii BA101 and in situ recovery by gas stripping,World Journal of Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard, A., andMargaritis, A., 2004, Empirical modeling of batch fermentation kineticsfor poly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

Recovery. The fermentation product(s) can be optionally recovered fromthe fermentation medium using any method known in the art including, butnot limited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Plants

The present invention also relates to plants, e.g., a transgenic plant,plant part, or plant cell, comprising a polynucleotide of the presentinvention so as to express and produce the variant in recoverablequantities. The variant may be recovered from the plant or plant part.Alternatively, the plant or plant part containing the variant may beused as such for improving the quality of a food or feed, e.g.,improving nutritional value, palatability, and rheological properties,or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a variant may beconstructed in accordance with methods known in the art. In short, theplant or plant cell is constructed by incorporating one or more(several) expression constructs encoding a variant into the plant hostgenome or chloroplast genome and propagating the resulting modifiedplant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a variant operably linked withappropriate regulatory sequences required for expression of thepolynucleotide in the plant or plant part of choice. Furthermore, theexpression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the variant is desired tobe expressed. For instance, the expression of the gene encoding avariant may be constitutive or inducible, or may be developmental, stageor tissue specific, and the gene product may be targeted to a specifictissue or plant part such as seeds or leaves. Regulatory sequences are,for example, described by Tague et al., 1988, Plant Physiol. 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay inducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a variant in the plant. For instance, the promoterenhancer element may be an intron that is placed between the promoterand the polynucleotide encoding a variant. For instance, Xu et al.,1993, supra, disclose the use of the first intron of the rice actin 1gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and can alsobe used for transforming monocots, although other transformation methodsare often used for these plants. Presently, the method of choice forgenerating transgenic monocots is particle bombardment (microscopic goldor tungsten particles coated with the transforming DNA) of embryoniccalli or developing embryos (Christou, 1992, Plant J. 2: 275-281;Shimamoto, 1994, Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992,Bio/Technology 10: 667-674). An alternative method for transformation ofmonocots is based on protoplast transformation as described by Omirullehet al., 1993, Plant Mol. Biol. 21: 415-428. Additional transformationmethods for use in accordance with the present disclosure include thosedescribed in U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which areherein incorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct prepared according to the present invention, transgenicplants may be made by crossing a plant having the construct to a secondplant lacking the construct. For example, a construct encoding a variantcan be introduced into a particular plant variety by crossing, withoutthe need for ever directly transforming a plant of that given variety.Therefore, the present invention encompasses not only a plant directlyregenerated from cells which have been transformed in accordance withthe present invention, but also the progeny of such plants. As usedherein, progeny may refer to the offspring of any generation of a parentplant prepared in accordance with the present invention. Such progenymay include a DNA construct prepared in accordance with the presentinvention, or a portion of a DNA construct prepared in accordance withthe present invention. Crossing results in the introduction of atransgene into a plant line by cross pollinating a starting line with adonor plant line. Non-limiting examples of such steps are furtherarticulated in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a variant ofthe present invention comprising: (a) cultivating a transgenic plant ora plant cell comprising a polynucleotide encoding the variant underconditions conducive for production of the variant; and (b) recoveringthe variant.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

Aspergillus fumigatus (NN051616) was used as the source of DNA encodingthe Family 6A cellobiohydrolase II. Aspergillus oryzae JaL355 (WO2002/40694) was used for expression of the Aspergillus fumigatuscellobiohydrolase II and variants thereof.

Media

PDA plates are composed of 39 grams of potato dextrose agar anddeionized water to 1 liter.

MDU2BP medium is composed of 45 g of maltose, 1 g of MgSO₄.7H₂O, 1 g ofNaCl, 2 g of K₂SO₄, 12 g of KH₂PO₄, 7 g of yeast extract, 2 g of urea,0.5 ml of AMG trace metals solution, and deionized water to 1 liter; pHto 5.0.

AMG trace metals solution is composed of 14.3 g of ZnSO₄.7H₂O, 2.5 g ofCuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g of FeSO₄.7H₂O, 8.5 g ofMnSO₄.H₂O, 3 g of citric acid, and deionized water to 1 liter.

LB medium is composed of 10 g of tryptone, 5 g of yeast extract, 5 g ofsodium chloride, and deionized water to 1 liter.

2×YT plates are composed of 16 g of tryptone, 10 g of yeast extract, 5 gof NaCl, 15 g of Noble agar, and deionized water to 1 liter.

Example 1 Aspergillus fumigatus Genomic DNA Extraction

Aspergillus fumigatus was grown in 250 ml of potato dextrose medium in abaffled shake flask at 37° C. and 240 rpm. Mycelia were harvested byfiltration, washed twice in 10 mM Tris-1 mM EDTA (TE) buffer (and frozenunder liquid nitrogen). Frozen mycelia were ground, by mortar andpestle, to a fine powder, which was resuspended in pH 8.0 buffercontaining 10 mM Tris, 100 mM EDTA, 1% Triton X-100, 0.5 Mguanidine-HCl, and 200 mM NaCl. DNase free RNase A was added at aconcentration of 20 mg/liter and the lysate was incubated at 37° C. for30 minutes. Cellular debris was removed by centrifugation, and DNA wasisolated by using a QIAGEN® Maxi 500 column (QIAGEN Inc., Valencia,Calif., USA). The columns were equilibrated in 10 ml of QBT washed with30 ml of QC, and eluted with 15 ml of QF (all buffers from QIAGEN Inc.,Valencia, Calif., USA). DNA was precipitated in isopropanol, washed in70% ethanol, and recovered by centrifugation. The DNA was resuspended inTE buffer.

Example 2 Construction of an Expression Vector for the Aspergillusfumigatus Family GH6A cellobiohydrolase II Gene

Two synthetic oligonucleotide primers shown below were designed to PCRamplify a full-length open reading frame of the Aspergillus fumigatusGH6A cellobiohydrolase II from genomic DNA. A TOPO Cloning Kit(Invitrogen, Carlsbad, Calif., USA) was used to clone the PCR product.An IN-FUSION™ Cloning Kit (BD Biosciences, Palo Alto, Calif., USA) wasused to clone the fragment into pAILo2 (WO 2004/099228).

In-Fusion Forward primer: (SEQ ID NO: 3)5′-ACTGGATTTACCATGAAGCACCTTGCATCTTCCATCG-3′ In-Fusion Reverse primer:(SEQ ID NO: 4) 5′-TCACCTCTAGTTAATTAAAAGGACGGGTTAGCGT-3′Bold letters represent coding sequence. The remaining sequence containssequence identity compared with the insertion sites of pAILo2.

Fifty picomoles of each of the primers above was used in a PCR reactioncontaining 500 ng of Aspergillus fumigatus genomic DNA, 1× ThermoPolreaction buffer (New England Biolabs, Ipswich, Mass., USA), 6 μl of a 10mM blend of dATP, dTTP, dGTP, and dCTP, and 0.1 units of Taq DNApolymerase (New England Biolabs, Ipswich, Mass., USA), in a final volumeof 50 μl. The amplification reaction was performed in an EPPENDORF®MASTERCYCLER® 5333 (Eppendorf Scientific, Inc., Westbury, N.Y., USA)programmed for one cycle at 98° C. for 2 minutes; and 35 cycles each at96° C. for 30 seconds, 61° C. for 30 seconds, and 72° C. for 2 minutes.After the 35 cycles, the reaction was incubated at 72° C. for 10 minutesand then cooled at 10° C. until further processed. To remove the A-tailsproduced by Taq DNA polymerase the reaction was incubated for 10 minutesat 68° C. in the presence of 1 unit of Pfx DNA polymerase (Invitrogen,Carlsbad, Calif., USA).

A 1.7 kb PCR reaction product was isolated using a 0.8% GTG-agarose gel(Cambrex Bioproducts, East Rutherford, N.J., USA) with 40 mM Trisbase-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer and 0.1 μg ofethidium bromide per ml. The DNA band was visualized with the aid of aDARK READER™ (Clare Chemical Research, Dolores, Colo., USA) to avoidUV-induced mutations. The 1.3 kb DNA band was excised from the gel witha disposable razor blade and purified using an ULTRAFREE®-DA spin cup(Millipore, Billerica, Mass., USA) according to the manufacturer'sinstructions.

The purified 1.7 kb PCR product was cloned into the vectorpCR®4Blunt-TOPO® (Invitrogen, Carlsbad, Calif., USA). Two microliters ofthe purified PCR product were mixed with 1 μl of a 2 M sodium chloridesolution and 1 μl of the TOPO® vector. The reaction was incubated atroom temperature for 15 minutes and then 2 μl of the reaction were usedto transform TOP10 E. coli competent cells according to themanufacturer's instructions (Invitrogen, Carlsbad, Calif., USA). Twoaliquots of 100 μl each of the transformation reaction were spread ontotwo 150 mm 2×YT plates supplemented with 100 μg of ampicillin per ml andincubated overnight at 37° C.

Eight recombinant colonies were each inoculated into 3 ml of LB mediumsupplemented with 100 μg of ampicillin per ml. Plasmid DNA was preparedfrom these cultures using a BIOROBOT® 9600 (QIAGEN Inc., Valencia,Calif., USA). Clones were analyzed by restriction digestion. Plasmid DNAfrom each clone was digested with the Eco RI and analyzed by 1% agarosegel electrophoresis using TAE buffer. Six out of eight clones had theexpected restriction digestion pattern. Clones 2, 4, 5, 6, 7 and 8 wereselected to be sequenced to confirm that there were no mutations in thecloned insert. Sequence analysis of their 5-prime and 3-prime endsindicated that clones 2, 6 and 7 had the correct sequence. These threeclones were selected for re-cloning into pAILo2. One microliter of eachclone was mixed with 17 μl of TE buffer diluted 10-fold and 1 μl of thismix was used to re-amplify the Aspergillus fumigatus GH6Acellobiohydrolase coding region.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 1 μl of the diluted mix of clones 2, 6 and 7, 1× PfxAmplification Buffer (Invitrogen, Carlsbad, Calif., USA), 6 μl of a 10mM blend of dATP, dTTP, dGTP, and dCTP, 2.5 units of PLATINUM® Pfx DNApolymerase (Invitrogen, Carlsbad, Calif., USA), and 1 μl of 50 mM MgSO₄,in a final volume of 50 μl. An EPPENDORF® MASTERCYCLER® 5333 was used toamplify the fragment programmed for one cycle at 98° C. for 2 minutes;and 35 cycles each at 94° C. for 30 seconds, 61° C. for 30 seconds, and68° C. for 1.5 minutes. After the 35 cycles, the reaction was incubatedat 68° C. for 10 minutes and then cooled at 10° C. until furtherprocessed. A 1.3 kb PCR reaction product was isolated using a 0.8%GTG-agarose gel with TAE buffer and 0.1 μg of ethidium bromide per ml.The DNA band was visualized with the aid of a DARK READER™ to avoidUV-induced mutations. The 1.7 kb DNA band was excised from the gel witha disposable razor blade and purified using an ULTRAFREE®-DA spin cupaccording to the manufacturer's instructions.

Vector pAILo2 was linearized by digestion with Nco I and Pac I. Thefragment was purified by agarose gel electrophoresis and ultrafiltrationas described above. Cloning of the purified PCR fragment into thelinearized and purified pAILo2 vector was performed using an IN-FUSION™Cloning Kit (BD Biosciences, Palo Alto, Calif., USA). The reaction (20μl) contained 1×IN-FUSION™ Buffer (BD Biosciences, Palo Alto, Calif.,USA), 1×BSA (BD Biosciences, Palo Alto, Calif., USA), 1 μl of IN-FUSION™enzyme (diluted 1:10) (BD Biosciences, Palo Alto, Calif., USA), 100 ngof pAILo2 digested with Nco I and Pac I, and 50 ng of the Aspergillusfumigatus purified 1.7 kb PCR product. The reaction was incubated atroom temperature for 30 minutes. A 2 μl sample of the reaction was usedto transform TOP10 E. coli competent cells according to themanufacturer's instructions. After a recovery period, two 100 μlaliquots from the transformation reaction were plated onto 150 mm 2×YTplates supplemented with 100 μg of ampicillin per ml. The plates wereincubated overnight at 37° C. Eight putative recombinant clones wasselected at random from the selection plates and plasmid DNA wasprepared from each one using a BIOROBOT® 9600. Clones were analyzed byPst I restriction digestion. Seven out of eight clones had the expectedrestriction digestion pattern. Clones 1, 2 and 3 were then sequenced toconfirm that there were no mutations in the cloned insert. Clone #2 wasselected and designated pAILo33.

Example 3 Construction of the Aspergillus fumigatus Family GH6Acellobiohydrolase II Gene Variants

Variants of the Aspergillus fumigatus GH6A cellobiohydrolase II wereconstructed by performing site-directed mutagenesis on pAILo33 (forpMaWo55, pMaWo 58, pMaWo78, and pAhyG90), or on pAhyG90 (for pMaWo70 andpMaWo71), pMaWo55 (for pMaWo72), pMaWo58 (for pAhyG108), pMaWo70 (forpMaWo73), or pMaWo71 (for pAhyG111) using a QUIKCHANGE® XL Site-DirectedMutagenesis Kit (Stratagene, La Jolla, Calif., USA). A summary of theoligos used for the site-directed mutagenesis and the variants obtainedare shown in Table 1.

The resulting variant plasmid DNAs were prepared using a BIOROBOT® 9600.Variant plasmid constructs were sequenced using a 3130xl GeneticAnalyzer (Applied Biosystems, Inc., Foster City, Calif., USA) to verifythe changes.

TABLE 1 Cloning Amino acid Primer Plasmid changes name Sequences NameD330N MaWo167 cctacacccagggaAaccccaactgcgacg pMaWo55 (SEQ ID NO: 5)MaWo168 cgtcgcagttggggtTtccctgggtgtagg (SEQ ID NO: 6) C254L MaWo173gagcgcctacctggagCTtgtcgactatgctctgaagc pMaWo58 (SEQ ID NO: 7) MaWo174gcttcagagcatagtcgacaAGctccaggtaggcgctc (SEQ ID NO: 8) M342F Af cel6a 3fgaagaagtacatcaacgccTtTgcgcctcttctcaaggaagccg pAhyG90 (SEQ ID NO: 9)Af cel6a 3r cggcttccttgagaagaggcgcAaAggcgttgatgtacttcttc (SEQ ID NO: 10)M342F + D330N MaWo167 cctacacccagggaAaccccaactgcgacg pMaWo70(SEQ ID NO: 5) MaWo168 cgtcgcagttggggtTtccctgggtgtagg (SEQ ID NO: 6)M342F + C254L MaWo173 gagcgcctacctggagCTtgtcgactatgctctgaagc pMaWo71(SEQ ID NO: 7) MaWo174 gcttcagagcatagtcgacaAGctccaggtaggcgctc(SEQ ID NO: 8) C254L + D330N MaWo173gagcgcctacctggagCTtgtcgactatgctctgaagc pMaWo72 (SEQ ID NO: 7) MaWo174gcttcagagcatagtcgacaAGctccaggtaggcgctc (SEQ ID NO: 8) C254L + D330N +MaWo173 gagcgcctacctggagCTtgtcgactatgctctgaagc pMaWo73 M342F(SEQ ID NO: 7) MaWo174 gcttcagagcatagtcgacaAGctccaggtaggcgctc(SEQ ID NO: 8) S360G + C254L MaWo181gcccacttcatcatggataccGGGcggaatggcgtccagccc pAhyG108 (SEQ ID NO: 11)MaWo182 gggctggacgccattccgCCCggtatccatgatgaagtgggc (SEQ ID NO: 12)S360G + C254L + MaWo181 gcccacttcatcatggataccGGGcggaatggcgtccagcccpAhyG111 M342F (SEQ ID NO: 11) MaWo182gggctggacgccattccgCCCggtatccatgatgaagtgggc (SEQ ID NO: 12) L285I + G286QMaWo205 ggctcggatggcccgccaacAtCCAAcccgccgcaacactcttcgcc pMaWo78(SEQ ID NO: 13) MaWo206 ggcgaagagtgttgcggcgggTTGGaTgttggcgggccatccgagcc(SEQ ID NO: 14)

Example 4 Expression of the Aspergillus fumigatus cDNA Encoding FamilyGH6A Cellobiohydrolase II Variants in Aspergillus oryzae JaL355

Aspergillus oryzae JaL355 protoplasts were prepared according to themethod of Christensen et al., 1988, Bio/Technology 6: 1419-1422. Five μgof expression vector (pMaWo55, pMaWo58, pAhyG90, pMaWo70, pMaWo71,pMaWo72, pMaWo73, pAhyG108, pAhyG111, or pMaWo78) was used to transformA. oryzae JaL355. Expression vector pAILo33 was transformed into A.oryzae JaL355 for expression of the Aspergillus fumigatus Family GH6Acellobiohydrolase II gene.

The transformation of A. oryzae JaL355 with pAILo33, pMaWo55, pMaWo58,pAhyG90, pMaWo70, pMaWo71, pMaWo72, pMaWo73, pAhyG108, pAhyG111, orpMaWo78 yielded about 1-10 transformants for each vector. Up to fourtransformants for each transformation were isolated to individual PDAplates.

Confluent PDA plates of the transformants were washed with 8 ml of 0.01%TWEEN® 20 and inoculated separately into 1 ml of MDU2BP medium insterile 24 well tissue culture plates and incubated at 34° C. Three daysafter incubation, 20 μl of harvested broth from each culture wasanalyzed using 8-16% Tris-Glycine SDS-PAGE gels (Invitrogen, Carlsbad,Calif., USA) according to the manufacturer's instructions. SDS-PAGEprofiles of the cultures showed that several transformants had a newmajor band of approximately 75 kDa.

A confluent plate of one transformant for each transformation (grown onPDA) was washed with 8 ml of 0.01% TWEEN® 20 and inoculated into 125 mlplastic shake flasks containing 25 ml of MDU2BP medium and incubated at34° C., either stationary or at 200 rpm to generate broth forcharacterization of the enzyme. The flasks were harvested on day 3 andfiltered using a 0.22 μm GP Express plus Membrane (Millipore, Bedford,Mass., USA).

Example 5 Measuring Thermostability of Aspergillus fumigatus Family GH6ACellobiohydrolase II Variants

Three ml of filtered broth for tested cultures from Example 4 weredesalted into 100 mM NaCl-50 mM sodium acetate pH 5.0 using ECONO-PAC®10DG Desalting Columns (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA). Protein in the desalted broths was concentrated to a 0.5 ml volumeusing a VIVASPIN® 6 (5 kDa cutoff) ultrafilter (Argos Technology, Elgin,Ill., USA).

The concentrated broths were diluted to 1 mg/ml protein concentrationusing 100 mM NaCl-50 mM sodium acetate pH 5.0. Two 25 μl aliquots ofeach 1 mg/ml protein sample were added to THERMOWELL® tube strip PCRtubes (Corning, Corning, N.Y., USA). One aliquot was kept on ice whilethe other aliquot was heated in an EPPENDORF® MASTERCYCLER® ep gradientS thermocycler (Eppendorf Scientific, Inc., Westbury, N.Y., USA) for 20minutes at 68° C. and then cooled to 4° C. before being placed on ice.Both samples were then diluted with 175 μl of 0.0114% TWEEN® 20-100 mMNaCl-50 mM sodium acetate pH 5.0.

Residual activity of the heated samples was then measured by determiningthe activity of the heated samples and the samples kept on ice inhydrolysis of phosphoric acid swollen cellulose (PASO). Ten microlitersof each sample was added in triplicate to a 96 well PCR plate(Eppendorf, Westbury, N.Y., USA). Then 190 μl of 2.1 g/l PASC in 0.01%TWEEN® 20-50 mM sodium acetate pH 5.0 buffer was added to the 10 μl ofsample and mixed. Glucose standards at 100, 75, 50, 25, 12.5 and 0 mgper liter in 50 mM sodium acetate pH 5.0 buffer were added in duplicateat 200 μl per well. The resulting mixtures were incubated for 30 minutesat 50° C. in an EPPENDORF® MASTERCYCLER® ep gradient S thermocycler. Thereactions were stopped by addition of 50 μl of 0.5 M NaOH to each well,including the glucose standards. The plate was then centrifuged in aSORVALL® RT 6000D centrifuge (Thermo Scientific, Waltham, Mass., USA)with a SORVALL® 1000B rotor equipped with a microplate carrier (ThermoScientific, Waltham, Mass., USA) for 2 minutes at 2,000 rpm.

Activity on PASC was determined by measuring reducing ends releasedduring a 30 minute hydrolysis at 50° C. One hundred microliters of eachsupernatant from the centrifuged plate was transferred to a separate96-well PCR plate. Fifty microliters of 1.5% (w/v) PHBAH(4-hydroxy-benzhydride, Sigma Chemical Co., St. Louis, Mo., USA) in 0.5M NaOH were added to each well. The plate was then heated in anEPPENDORF® MASTERCYCLER® ep gradient S thermocycler at 95° C. for 15minutes and then 15° C. for 5 minutes. A total of 100 μl of each samplewas transferred to a clear, flat-bottom 96-well plate (Corning, Inc.,Corning, N.Y., USA). The absorbance at 410 nm was then measured using aSPECTRAMAX® 340 pc spectrophotometric plate reader (Molecular Devices,Sunnyvale, Calif., USA). The concentration of reducing ends released wasdetermined from a straight-line fit to the concentration of reducingends released versus the absorbance at 410 nm for the glucose standards.Residual activity was then calculated by dividing the reducing endsreleased from PASC hydrolyzed by a heated sample by the reducing endsreleased from PASC hydrolyzed by a sample that was kept on ice. Activityof the cellobiohydrolase II variants was compared to activity of theparent enzyme.

The results shown in FIGS. 1A and 1B demonstrated an increase inthermostability by a higher residual activity for indicated variantscompared to the parent enzyme.

Example 6 Measuring Hydrolytic Activity of Aspergillus fumigatus FamilyGH6A Cellobiohydrolase II Variants

One hundred fifty ml of broth supernatant containing Aspergillusfumigatus Family GH6A cellobiohydrolase II wild-type or variantspAhyG108, pAhyG111 or pMaWo73, were concentrated using a 10 kDa cut-offVivaspin ultra-filter (Millipore, Billerica, Mass., USA). Up to 13 ml ofconcentrated samples were loaded on a HiLoad® 26/60 Superdex 200 column(GE Healthcare Life Sciences, Piscataway, N.J., USA) equilibrated with100 mM NaCl-20 mM sodium acetate pH 5.0 buffer. Protein was eluted with100 mM sodium chloride-20 mM sodium acetate pH 5.0 buffer. Fractions of7 ml were collected for each sample and submitted to SDS-PAGE analysison a 8-16% Bior-Rad Criterion stain-free Tris-HCl gel (Bio-RadLaboratories, Inc., Hercules, Calif., USA). Fractions that eluted at 230to 240 ml were pooled and showed greater than 95% purity.

The purified samples were diluted to 125, 112.5, 101.3, 91.1, 82.0, 73.8and 66.4 μg/ml protein concentrations using 50 mM sodium acetate pH 5.0.Hydrolytic activity of the samples was then measured by hydrolyzingphosphoric acid swollen cellulose (PASC). Ten microliters of each samplewas added in triplicate to a 96 well PCR plate (Eppendorf, Westbury,N.Y., USA). Then 190 μl of 2.1 g/l PASC in 0.01% TWEEN® 20-50 mM sodiumacetate pH 5.0 buffer was added to the 10 μl of sample and mixed.Glucose standards at 100, 75, 50, 25, 12.5 and 0 mg per liter in 50 mMsodium acetate pH 5.0 buffer were added in duplicate at 200 μl per well.The resulting mixtures were incubated for 30 minutes at 50° C. in anEPPENDORF®MASTERCYCLER® ep gradient S thermocycler. The reactions werestopped by addition of 50 μl of 0.5 M NaOH to each well, including theglucose standards. The plate was then centrifuged in a SORVALL® RT 6000Dcentrifuge (Thermo Scientific, Waltham, Mass., USA) with a SORVALL®1000B rotor equipped with a microplate carrier (Thermo Scientific,Waltham, Mass., USA) for 2 minutes at 2,000 rpm.

Activity on PASC was determined by measuring reducing ends releasedduring a 30 minute hydrolysis at 50° C. One hundred microliters of eachsupernatant from the centrifuged plate was transferred to a separate96-well PCR plate. Fifty microliters of 1.5% (w/v) PHBAH(4-hydroxy-benzhydride, Sigma Chemical Co., St. Louis, Mo., USA) in 0.5M NaOH were added to each well. The plate was then heated in anEPPENDORF® MASTERCYCLER® ep gradient S thermocycler at 95° C. for 15minutes and then 15° C. for 5 minutes. A total of 100 μl of each samplewas transferred to a clear, flat-bottom 96-well plate (Corning, Inc.,Corning, N.Y., USA). The absorbance at 410 nm was then measured using aSPECTRAMAX® 340 pc spectrophotometric plate reader (Molecular Devices,Sunnyvale, Calif., USA). The concentration of reducing ends released wasdetermined from a straight-line fit to the concentration of reducingends released versus the absorbance at 410 nm for the glucose standards.Activity of the cellobiohydrolase II variants was compared to activityof the parent enzyme.

The results shown in FIG. 2 demonstrated an increase in hydrolyticactivity of each variant containing the S360G mutation compared to theparent enzyme.

The present invention may be further described by the following numberedparagraphs:

[1] An isolated variant of a parent cellobiohydrolase, comprising asubstitution at one or more (several) positions corresponding topositions 254, 285, 286, 330, 342, and 360 of SEQ ID NO: 2, wherein thevariant has cellobiohydrolase activity.

[2] The variant of paragraph 1, wherein the parent cellobiohydrolase is:

a. a polypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 2;

b. a polypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions with (i) the mature polypeptide genomic codingsequence of SEQ ID NO: 1, (ii) the mature polypeptide cDNA codingsequence contained in SEQ ID NO: 1, or (iii) the full-lengthcomplementary strand of (i) or (ii);

c. a polypeptide encoded by a polynucleotide having at least 60%identity to the mature polypeptide genomic coding sequence of SEQ ID NO:1, or the mature polypeptide cDNA coding sequence contained in SEQ IDNO: 1;

d. a fragment of the mature polypeptide of SEQ ID NO: 2, which hascellobiohydrolase activity.

[3] The variant of paragraph 1 or 2, wherein the parentcellobiohydrolase has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 2.

[4] The variant of any one of paragraphs 1-3, wherein the parentcellobiohydrolase is encoded by a polynucleotide that hybridizes underlow stringency conditions with (i) the mature polypeptide genomic codingsequence of SEQ ID NO: 1, (ii) the mature polypeptide cDNA codingsequence contained in SEQ ID NO: 1, or (iii) the full-lengthcomplementary strand of (i) or (ii).

[5] The variant of any one of paragraphs 1-4, wherein the parentcellobiohydrolase is encoded by a polynucleotide having at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to the mature polypeptidegenomic coding sequence of SEQ ID NO: 1, or the mature polypeptide cDNAcoding sequence contained in SEQ ID NO: 1.

[6] The variant of any one of paragraphs 1-5, wherein the parentcellobiohydrolase comprises or consists of the mature polypeptide of SEQID NO: 2.

[7] The variant of any one of paragraphs 1-5, wherein the parentcellobiohydrolase is a fragment of the mature polypeptide of SEQ ID NO:2, wherein the fragment has cellobiohydrolase activity.

[8] The variant of any one of paragraphs 1-7, which has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95% identity, at least 96%, at least 97%, atleast 98%, at least 99%, but less than 100%, sequence identity to theamino acid sequence of the parent cellobiohydrolase.

[9] The variant of any one of paragraphs 1-8, which has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, and at least 99%, but less than 100% sequence identity to themature polypeptide of SEQ ID NO: 2.

[10] The variant of any one of paragraphs 1-9, wherein the variantcomprises at least 370 amino acid residues, e.g., at least 390 or atleast 410 amino acid residues.

[11] The variant of any one of paragraphs 1-10, wherein the variantcomprises 1-5 substitutions, such as 1, 2, 3, 4, or 5 substitutions.

[12] The variant of any one of paragraphs 1-11, comprising asubstitution at a position corresponding to position 254 of SEQ ID NO:2.

[13] The variant of paragraph 12, wherein the substitution is with Leu.

[14] The variant of any one of paragraphs 1-13, comprising asubstitution at a position corresponding to position 330 of SEQ ID NO:2.

[15] The variant of paragraph 14, wherein the substitution is with Asn.

[16] The variant of any one of paragraphs 1-15, comprising asubstitution at a position corresponding to position 342 of SEQ ID NO:2.

[17] The variant of paragraph 16, wherein the substitution is with Phe.

[18] The variant of any one of paragraphs 1-17, comprising asubstitution at a position corresponding to position 360 of SEQ ID NO:2.

[19] The variant of paragraph 18, wherein the substitution is with Gly.

[20] The variant of any one of paragraphs 1-19, comprising asubstitution at a position corresponding to position 285 of SEQ ID NO:2.

[21] The variant of paragraph 20, wherein the substitution is with Ile.

[22] The variant of any one of paragraphs 1-21, comprising asubstitution at a position corresponding to position 286 of SEQ ID NO:2.

[23] The variant of paragraph 22, wherein the substitution is with Gln.

[24] The variant of any one of paragraphs 1-23, comprising substitutionsat least two positions corresponding to any of positions 254, 285, 286,330, 342, and 360 of SEQ ID NO: 2.

[25] The variant of any one of paragraphs 1-23, comprising substitutionsat least three positions corresponding to any of positions 254, 285,286, 330, 342, and 360 of SEQ ID NO: 2.

[26] The variant of any one of paragraphs 1-23, comprising substitutionsat least four positions corresponding to any of positions 254, 285, 286,330, 342, and 360 of SEQ ID NO: 2.

[27] The variant of any one of paragraphs 1-23, comprising substitutionsat least five positions corresponding to any of positions 254, 285, 286,330, 342, and 360 of SEQ ID NO: 2.

[27] The variant of any one of paragraphs 1-23, comprising substitutionsat each position corresponding to any of positions 254, 285, 286, 330,342, and 360 of SEQ ID NO: 2.

[28] The variant of any one of paragraphs 1-27, comprising one or more(several) substitutions selected from the group consisting of C254L,L285I, G286Q, D330N, M342F, and S360G.

[29] The variant of paragraph 28, comprising the substitution C254L.

[30] The variant of paragraph 28, comprising the substitution L285I.

[31] The variant of paragraph 28, comprising the substitution G286Q.

[32] The variant of paragraph 28, comprising the substitution D330N.

[33] The variant of paragraph 28, comprising the substitution M342F.

[34] The variant of paragraph 28, comprising the substitution S360G.

[35] The variant of paragraph 28, comprising two substitutions selectedfrom the group consisting of C254L, L285I, G286Q, D330N, M342F, andS360G.

[36] The variant of paragraph 35, comprising the substitutionsC254L+L285I.

[37] The variant of paragraph 35, comprising the substitutionsC254L+G286Q.

[38] The variant of paragraph 35, comprising the substitutionsC254L+D330N.

[39] The variant of paragraph 35, comprising the substitutionsC254L+M342F.

[40] The variant of paragraph 35, comprising the substitutionsC254L+S360G.

[41] The variant of paragraph 35, comprising the substitutionsL285I+G286Q.

[42] The variant of paragraph 35, comprising the substitutionsL285I+D330N.

[43] The variant of paragraph 35, comprising the substitutionsL285I+M342F.

[44] The variant of paragraph 35, comprising the substitutionsL285I+S360G.

[45] The variant of paragraph 35, comprising the substitutionsG286Q+D330N.

[46] The variant of paragraph 35, comprising the substitutionsG286Q+M342F.

[47] The variant of paragraph 35, comprising the substitutionsG286Q+S360G.

[48] The variant of paragraph 28, comprising three substitutionsselected from the group consisting of C254L, L285I, G286Q, D330N, M342F,and S360G.

[49] The variant of paragraph 48, comprising the substitutionsC254L+L285I+G286Q.

[50] The variant of paragraph 48, comprising the substitutionsC254L+L285I+D330N.

[51] The variant of paragraph 48, comprising the substitutionsC254L+L285I+M342F.

[52] The variant of paragraph 48, comprising the substitutionsC254L+L285I+S360G.

[53] The variant of paragraph 48, comprising the substitutionsC254L+G286Q+D330N.

[54] The variant of paragraph 48, comprising the substitutionsC254L+G286Q+M342F.

[55] The variant of paragraph 48, comprising the substitutionsC254L+G286Q+S360G.

[56] The variant of paragraph 48, comprising the substitutionsC254L+D330N+M342F.

[57] The variant of paragraph 48, comprising the substitutionsC254L+D330N+S360G.

[58] The variant of paragraph 48, comprising the substitutionsC254L+M342F+S360G.

[59] The variant of paragraph 48, comprising the substitutionsL285I+G286Q+D330N.

[60] The variant of paragraph 48, comprising the substitutionsL285I+G286Q+M342F.

[61] The variant of paragraph 48, comprising the substitutionsL285I+G286Q+S360G.

[62] The variant of paragraph 48, comprising the substitutionsL285I+D330N+M342F.

[63] The variant of paragraph 48, comprising the substitutionsL285I+D330N+S360G.

[64] The variant of paragraph 48, comprising the substitutionsL285I+M342F+S360G.

[65] The variant of paragraph 48, comprising the substitutionsG286Q+D330N+M342F.

[66] The variant of paragraph 48, comprising the substitutionsG286Q+D330N+S360G.

[67] The variant of paragraph 48, comprising the substitutionsG286Q+M342F+S360G.

[68] The variant of paragraph 48, comprising the substitutionsD330N+M342F+S360G.

[69] The variant of paragraph 28, comprising four substitutions selectedfrom the group consisting of C254L, L285I, G286Q, D330N, M342F, andS360G.

[70] The variant of paragraph 69, comprising the substitutionsC254L+L285I+G286Q+D330N.

[71] The variant of paragraph 69, comprising the substitutionsC254L+L285I+G286Q+M342F.

[72] The variant of paragraph 69, comprising the substitutionsC254L+L285I+D330N+M342F.

[73] The variant of paragraph 69, comprising the substitutionsC254L+G286Q+D330N+M342F.

[74] The variant of paragraph 69, comprising the substitutionsL285I+G286Q+D330N+M342F.

[75] The variant of paragraph 69, comprising the substitutionsC254L+L285I+G286Q+S360G.

[76] The variant of paragraph 69, comprising the substitutionsC254L+L285I+D330N+S360G.

[77] The variant of paragraph 69, comprising the substitutionsC254L+G286Q+D330N+S360G.

[78] The variant of paragraph 69, comprising the substitutionsL285I+G286Q+D330N+S360G.

[79] The variant of paragraph 69, comprising the substitutionsC254L+L285I+M342F+S360G.

[80] The variant of paragraph 69, comprising the substitutionsC254L+G286Q+M342F+S360G.

[81] The variant of paragraph 69, comprising the substitutionsL285I+G286Q+M342F+S360G.

[82] The variant of paragraph 69, comprising the substitutionsC254L+D330N+M342F+S360G.

[83] The variant of paragraph 69, comprising the substitutionsL285I+D330N+M342F+S360G.

[84] The variant of paragraph 69, comprising the substitutionsG286Q+D330N+M342F+S360G.

[85] The variant of paragraph 28, comprising five substitutions selectedfrom the group consisting of C254L, L285I, G286Q, D330N, M342F, andS360G.

[86] The variant of paragraph 85, comprising the substitutionsL285I+G286Q+D330N+M342F+S360G.

[87] The variant of paragraph 85, comprising the substitutionsC254L+G286Q+D330N+M342F+S360G.

[88] The variant of paragraph 85, comprising the substitutionsC254L+L285I+D330N+M342F+S360G.

[89] The variant of paragraph 85, comprising the substitutionsC254L+L285I+G286Q+M342F+S360G.

[90] The variant of paragraph 85, comprising the substitutionsC254L+L285I+G286Q+D330N+S360G.

[91] The variant of paragraph 85, comprising the substitutionsC254L+L285I+G286Q+D330N+M342F.

[92] The variant of paragraph 28, comprising the substitutionsC254L+L285I+G286Q+D330N+M342F+S360G.

[93] The variant of any one of paragraphs 1-92, further comprising oneor more (several) substitutions at any position corresponding topositions 245, 382, 420, 437, and 440 of SEQ ID NO: 2.

[94] The variant of paragraph 93, comprising two substitutions at anyposition corresponding to positions 245, 382, 420, 437, and 440 of SEQID NO: 2.

[95] The variant of paragraph 93, comprising three substitutions at anyposition corresponding to positions 245, 382, 420, 437, and 440 of SEQID NO: 2.

[96] The variant of paragraph 93, comprising four substitutions at anyposition corresponding to positions 245, 382, 420, 437, and 440 of SEQID NO: 2.

[97] The variant of paragraph 93, comprising substitutions at eachposition corresponding to positions 245, 382, 420, 437, and 440 of SEQID NO: 2.

[98] The variant of any one of paragraphs 93-97, comprising asubstitution at a position corresponding to position 245 of SEQ ID NO:2.

[99] The variant of paragraph 98, wherein the substitution is Ser.

[100] The variant of any one of paragraphs 93-99, comprising asubstitution at a position corresponding to position 382 of SEQ ID NO:2.

[101] The variant of paragraph 100, wherein the substitution is Cys.

[102] The variant of any one of paragraphs 93-101, comprising asubstitution at a position corresponding to position 420 of SEQ ID NO:2.

[103] The variant of paragraph 102, wherein the substitution is Ile.

[104] The variant of any one of paragraphs 93-103, comprising asubstitution at a position corresponding to position 437 of SEQ ID NO:2.

[105] The variant of paragraph 104, wherein the substitution is Gln.

[106] The variant of any one of paragraphs 93-105, comprising asubstitution at a position corresponding to position 440 of SEQ ID NO:2.

[107] The variant of paragraph 106, wherein the substitution is Cys.

[108] The variant of any one of paragraphs 93-107, comprising one ormore (several) substitutions selected from the group consisting ofA245S, G382C, L420I, T437Q, and Q440C.

[109] The variant of paragraph 108, comprising two substitutionsselected from the group consisting of A245S, G382C, L420I, T437Q, andQ440C.

[110] The variant of paragraph 109, comprising the substitutionsL420I+T437Q.

[111] The variant of paragraph 109, comprising the substitutionsA245S+L420I.

[112] The variant of paragraph 109, comprising the substitutionsG382C+L420I.

[113] The variant of paragraph 109, comprising the substitutionsL420I+Q440C.

[114] The variant of paragraph 109, comprising the substitutionsA245S+T437Q.

[115] The variant of paragraph 109, comprising the substitutionsG382C+T437Q.

[116] The variant of paragraph 109, comprising the substitutionsT437Q+Q440C.

[117] The variant of paragraph 109, comprising the substitutionsA245S+G382C.

[118] The variant of paragraph 109, comprising the substitutionsA245S+Q440C.

[119] The variant of paragraph 109, comprising the substitutionsG382C+Q440C.

[120] The variant of paragraph 108, comprising three substitutionsselected from the group consisting of A245S, G382C, L420I, T437Q, andQ440C.

[121] The variant of paragraph 120, comprising the substitutionsA245S+L420I+T437Q.

[122] The variant of paragraph 120, comprising the substitutionsG382C+L420I+T437Q.

[123] The variant of paragraph 120, comprising the substitutionsL420I+T437Q+Q440C.

[124] The variant of paragraph 120, comprising the substitutionsA245S+G382C+L420I.

[125] The variant of paragraph 120, comprising the substitutionsA245S+L420I+Q440C.

[126] The variant of paragraph 120, comprising the substitutionsG382C+L420I+Q440C.

[127] The variant of paragraph 120, comprising the substitutionsA245S+G382C+T437Q.

[128] The variant of paragraph 120, comprising the substitutionsA245S+T437Q+Q440C.

[129] The variant of paragraph 120, comprising the substitutionsG382C+T437Q+Q440C.

[130] The variant of paragraph 120, comprising the substitutionsA245S+G382C+Q440C.

[131] The variant of paragraph 108, comprising four substitutionsselected from the group consisting of A245S, G382C, L420I, T437Q, andQ440C.

[132] The variant of paragraph 131, comprising the substitutionsA245S+G382C+T437Q+Q440C.

[133] The variant of paragraph 131, comprising the substitutionsA245S+G382C+L420I+Q440C.

[134] The variant of paragraph 131, comprising the substitutionsG382C+L420I+T437Q+Q440C.

[135] The variant of paragraph 131, comprising the substitutionsA245S+L420I+T437Q+Q440C.

[136] The variant of paragraph 131, comprising the substitutionsA245S+G382C+L420I+T437Q.

[137] The variant of paragraph 108, comprising the substitutionsA245S+G382C+L420I+T437Q+Q440C.

[138] The variant of any one of paragraphs 1-137, which has improvedthermostability relative to the parent cellobiohydrolase.

[139] The variant of any one of paragraphs 1-138, wherein the maturepolypeptide is amino acids 20-454 of SEQ ID NO:2.

[140] The variant of any one of paragraphs 1-139, wherein the maturepolypeptide coding sequence is nucleotides 58-1710 of SEQ ID NO: 1.

[141] An isolated polynucleotide encoding the variant of any one ofparagraphs 1-140.

[142] A nucleic acid construct comprising the polynucleotide ofparagraph 141.

[143] An expression vector comprising the polynucleotide of paragraph141.

[144] A host cell comprising the polynucleotide of paragraph 141.

[145] A method of producing a variant of a parent cellobiohydrolase,comprising:

(a) cultivating the host cell of paragraph 144 under conditions suitablefor the expression of the variant; and

(b) recovering the variant.

[146] A transgenic plant, plant part or plant cell transformed with thepolynucleotide of paragraph 141.

[147] A method of producing a variant of any one of paragraphs 1-140,comprising:

(a) cultivating a transgenic plant or a plant cell comprising apolynucleotide encoding the variant under conditions conducive forproduction of the variant; and

(b) recovering the variant.

[148] A method for obtaining the variant of any one of paragraphs 1-140,comprising:

(a) introducing into a parent cellobiohydrolase a substitution at one ormore (several) positions corresponding to positions 254, 285, 286, 330,342, and 360 of the mature polypeptide of SEQ ID NO: 2, wherein thevariant has cellobiohydrolase activity; and

(b) recovering the variant.

[149] The method of paragraph 148 further comprising introducing intothe parent cellobiohydrolase a substitution at one or more (several)positions corresponding to positions 245, 382, 420, 437, and 440 of themature polypeptide of SEQ ID NO: 2

[150] An enzyme composition comprising the variant of any one ofparagraphs 1-140.

[151] The enzyme composition of paragraph 150, further comprising acellulolytic enzyme selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[152] The enzyme composition of paragraph 150 or 151, further comprisinga polypeptide having cellulolytic enhancing activity.

[153] The enzyme composition of any one of paragraphs 150-152, furthercomprising an enzyme selected from the group consisting of a xylanase, ahemicellulase, an esterase, a protease, a laccase, and a peroxidase.

[154] A method for degrading or converting a cellulosic material,comprising: treating the cellulosic material with an enzyme compositionof any one of paragraphs 150-153.

[155] The method of paragraph 154, wherein the cellulosic material ispretreated.

[156] The method of paragraph 154 or 155, further comprising recoveringthe degraded cellulosic material.

[157] The method of paragraph 156, wherein the degraded cellulosicmaterial is a sugar.

[158] The method of paragraph 157, wherein the sugar is selected fromthe group consisting of glucose, xylose, mannose, galactose, andarabinose.

[159] A method for producing a fermentation product from cellulosicmaterial, comprising:

(a) saccharifying the cellulosic material with an enzyme composition ofany of claims 150-153 to produce saccharified cellulosic material;

(b) fermenting the saccharified cellulosic material with one or morefermenting microorganisms to produce a fermentation product; and

(c) recovering the fermentation product.

[160] The method of paragraph 159, wherein the cellulosic material ispretreated.

[161] The method of paragraph 159 or 160, wherein steps (a) and (b) areperformed simultaneously.

[162] The method of any one of paragraphs 159-161, wherein thefermentation product is an alcohol, an organic acid, a ketone, an aminoacid, or a gas.

[163] A method of fermenting a cellulosic material, comprising:fermenting the cellulosic material with one or more fermentingmicroorganisms, wherein the cellulosic material is saccharified with anenzyme composition of any one of paragraphs 150-153.

[164] The method of paragraph 163, wherein the fermenting of thecellulosic material produces a fermentation product.

[165] The method of paragraph 163 or 164, further comprising recoveringthe fermentation product.

[166] The method of any one of paragraphs 163-165, wherein thecellulosic material is pretreated before being saccharified.

[167] The method of any one of paragraphs 163-166, wherein thefermentation product is an alcohol, an organic acid, a ketone, an aminoacid, or a gas.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

What is claimed is:
 1. An isolated variant of a parent cellobiohydrolase, comprising a substitution at one or more positions corresponding to positions 254, 285, 286, 330, 342, and 360 of SEQ ID NO: 2, wherein the variant has cellobiohydrolase activity and wherein the parent cellobiohydrolase is selected from the group consisting of: (a) a polypeptide having at least 90% sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizes under very high stringency conditions with (i) the mature polypeptide genomic coding sequence of SEQ ID NO: 1, (ii) the mature polypeptide cDNA coding sequence contained in SEQ ID NO: 1, or (iii) the full-length complementary strand of (i) or (ii), wherein very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.; (c) a polypeptide encoded by a polynucleotide having at least 90% sequence identity to the mature polypeptide genomic coding sequence of SEQ ID NO: 1, or the mature polypeptide cDNA coding sequence contained in SEQ ID NO: 1; and (d) a fragment of the mature polypeptide of SEQ ID NO: 2, which has cellobiohydrolase activity.
 2. The variant of claim 1, wherein the parent cellobiohydrolase comprises or consists of the mature polypeptide of SEQ ID NO:
 2. 3. The variant of claim 1, wherein the substitution corresponding to position 254 is with Leu, the substitution corresponding to position 285 is with Ile, the substitution corresponding to position 286 is with Gln, the substitution corresponding to position 330 is with Asn, the substitution corresponding to position 342 is with Phe, and the substitution corresponding to position 360 is with Gly.
 4. The variant of claim 1, wherein the substitution corresponding to position 254 is C254L, the substitution corresponding to position 285 is L285I, the substitution corresponding to position 286 is G286Q, the substitution corresponding to position 330 is D330N, the substitution corresponding to position 342 is M342F, and the substitution corresponding to position 360 is S360G.
 5. The variant of claim 1, further comprising a substitution at one or more positions corresponding to positions 245, 382, 420, 437, and 440 of SEQ ID NO:
 2. 6. The variant of claim 5, wherein the substitution corresponding to position corresponding to position 245 is with Ser, the substitution corresponding to position 382 is with Cys, the substitution corresponding to position 420 is with Ile, the substitution corresponding to position 437 is with Gln, and the substitution corresponding to position 440 is with Cys.
 7. The variant of claim 5, wherein the substitution corresponding to position corresponding to position 245 is A245S, the substitution corresponding to position 382 is G382C, the substitution corresponding to position 420 is L420I, the substitution corresponding to position 437 is T437Q, and the substitution corresponding to position 440 is Q440C.
 8. The variant of claim 1, which has improved thermostability relative to the parent cellobiohydrolase.
 9. The variant of claim 1, wherein the mature polypeptide of SEQ ID NO: 2 is amino acids 20-454 of SEQ ID NO:
 2. 10. The variant of claim 1, wherein the mature polypeptide coding sequence of SEQ ID NO: 1 is nucleotides 58-1710 of SEQ ID NO:
 1. 11. An isolated polynucleotide encoding the variant of claim
 1. 12. A nucleic acid construct comprising the polynucleotide of claim
 11. 13. An expression vector comprising the polynucleotide of claim
 11. 14. An isolated host cell comprising the polynucleotide of claim
 11. 15. A method of producing a variant of a parent cellobiohydrolase, comprising: (a) cultivating the host cell of claim 14 under conditions suitable for the expression of the variant; and (b) recovering the variant.
 16. A method for obtaining the variant of claim 1, comprising: (a) introducing into a parent cellobiohydrolase a substitution at one or more positions corresponding to positions 254, 285, 286, 330, 342, and 360 of SEQ ID NO: 2; and (b) recovering the variant.
 17. An enzyme composition comprising the variant of claim
 1. 18. The enzyme composition of claim 17, further comprising a cellulolytic enzyme selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase, and optionally further comprises a polypeptide having cellulolytic enhancing activity, a xylanase, a hemicellulase, an esterase, a protease, a laccase, or a peroxidase.
 19. A method for degrading or converting a cellulosic material, comprising: treating the cellulosic material with an enzyme composition of claim
 17. 20. A method for producing a fermentation product from cellulosic material, comprising: (a) saccharifying the cellulosic material with an enzyme composition of claim 17 to produce saccharified cellulosic material; (b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to produce a fermentation product; and (c) recovering the fermentation product.
 21. A method of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition of claim
 17. 22. The variant of claim 1, wherein the parent cellobiohydrolase is a polypeptide having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:
 2. 23. The variant of claim 1, wherein the parent cellobiohydrolase is encoded by a polynucleotide having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide genomic coding sequence of SEQ ID NO: 1, or the mature polypeptide cDNA coding sequence contained in SEQ ID NO:
 1. 